Il-36r signal-specific inhibitory antibody

Abstract

Provided is an IL-36R signal-specific inhibitory antibody that is an IL-36R neutralizing antibody specifically binding to human IL-36R and inhibiting the signals of IL-36R agonists and that shows little or no inhibition of the signal of IL-36[beta] from among IL-36[alpha], IL-36[beta] and IL-36[gamma]. This antibody shows a

Description

[Technical Field]

[0001] This invention relates to antibodies that specifically block IL-36R signaling. Specifically, it relates to antibodies that bind to IL-36R in vertebrates and block IL-36R signaling. [Previous Technology]

[0002] <IL-36R>

[0003] Cytokines are small molecular weight (generally 5-30 kDa) polypeptides that possess physiological activity even in trace amounts. They are a general term for proteins secreted by cells that affect the behavior of neighboring cells with corresponding receptors. Known examples include interleukin (IL), interferon (IFN), tumor necrosis factor (TNF), and chemokines. With the progress of research, their classification names have been published by the Human Genome Nomenclature Committee (HGNC) of the Human Genome Organization (HUGO). Interleukin is a substance secreted by lymphocytes or macrophages that regulates the immune system and is responsible for intercellular communication. It is classified according to the orientation of its ligand or receptor (Non-Patent Literature 1).

[0004] The interleukin-36 receptor (IL-36R) and its ligands were systematically reported from the 1990s to the first half of the 2000s by the Human Genome Project as gene sequences similar to the IL-1 family. Since its discovery, IL-36R has been known to possess a TIR domain in an intracellular region, similar to the TNF receptor or Toll-like receptors. Subsequently, based on the distribution of the expression in the tissue, it was confirmed that the signal cascade associated with MyD88 (myeloid differentiation primary response gene 88) is activated through the common helper protein (IL-1RAcP) in the IL-1 family receptors, thereby activating the signaling of nuclear factor-kappa B (NFkB), mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK), and its importance in the immune response was recognized (Non-Patent Literature 2). IL-36R is most commonly found in the skin, thymus, and mucous membranes. Furthermore, IL-36R has been observed in a variety of immune-active cells, including keratinocytes (non-patent literature 3, non-patent literature 4), dendritic cells (non-patent literature 3, 5, 9), monocytes (non-patent literature 3, 5), fibroblasts (non-patent literature 3), neutrophils (non-patent literature 3, 7), T cells (non-patent literature 8), macrophages (non-patent literature 3, 6), and B cells (non-patent literature 8).

[0005] IL-1 family receptors have low sequence similarity in ligand binding sites, but their structures are similar. The extracellular region is a glycoprotein consisting of three domains formed by two β-sheets, called the Ig-like domain, and the glycan modification sites differ between families. Like other IL-1 family receptors, the IL-36 receptor (hereinafter appropriately referred to as "IL-36R") is presumed to form a trimer with the agonist ligand and the IL-1 receptor accessory protein (IL-1RAcP), as experimentally demonstrated (Non-Patent Literature 10).

[0006] From the perspective of ligands, IL-36, like IL-1, is known to have both agonists and antagonists. As ligands, three agonists (IL-36α, IL-36β, IL-36γ) and two antagonists (IL-36Ra, IL-38) have been reported (Non-Patent Literature 11). Like other members of the IL-1 family, the various ligands of IL-36R are usually located intracellularly. While the secretion mechanism is unclear, it is believed that they are released extracellularly due to cell death or an immune response, and activated by N-terminal cleavage by proteases. IL-36 agonists, through processing at appropriate sites, exhibit a 980-fold to 36000-fold increase in binding activity to the IL-36R / IL-1RAcP complex, and a 1500-fold to 8000-fold increase in biological activity as obtained by reporter assay (Non-Patent Literature 15). Unlike IL-1α, IL-1β, or IL-18, which are activated by apoptotic proteases, IL-36 is activated by autolysin-S or neutrophil elastase (Non-Patent Document 12). The activating protease varies depending on the ligand, and its activity also varies depending on the activating enzyme. For example, reports indicate that IL-36γ is more potent than cathepsin G due to neutrophil elastase (Non-Patent Document 13). Inhibitors targeting the enzymes activating the ligands have been developed (Non-Patent Document 14). However, it is unclear whether all reported enzymes are present. For example, IL-18, which is reported to be elevated in atopic dermatitis, is reported to be activated by enzymes such as rennet derived from obese cells, granzyme B derived from CD8+ T cells, and Sp-A derived from Staphylococcus aureus. Therefore, it is not surprising that IL-36 is also activated by enzymes from indigenous bacteria.

[0007] Regarding IL-36Ra, similar to the IL-36R agonist, processing is crucial. Reports indicate that the removal of the N-terminal methionine is important for its activity (Non-Patent Literature 15). Regarding the removal of the N-terminal methionine in eukaryotes, reports indicate that the amino acid sequence following the N-terminal methionine is important (Non-Patent Literature 16). Since the amino acid following the N-terminal methionine in IL-36Ra is Val, it is highly likely to be processed during translation. In recent years, reports have indicated that neutrophil elastase, particularly the enzyme that activates IL-36γ, activates IL-36Ra (Non-Patent Literature 17), suggesting that the balance between IL-36 agonists and antagonists is under complex control. Regarding another antagonist, IL-38, although reports exist, the activated form is not yet commercially available, and its potential binding to other IL-1 family receptors is still under investigation. However, reports indicate that when full-length IL-38 is administered to dysplastic psoriasis model animals, inflammatory cytokines such as IL-17 or IL-22 are reduced, which is also related to the effectiveness of anti-IL-17 antibodies in actual clinical practice (Non-Patent Literature 18).

[0008] IL-36 antagonists are thought to inhibit or regulate IL-36R signaling by binding to receptors or competing with ligands. According to reports on the processing of IL-36 ligands and IL-36Ra, IL-36Ra loses its ability to block IL-36R signaling by not removing the N-terminal methionine 1 residue (Non-Patent Literature 15). <The Association Between IL-36R and Psoriasis>

[0009] According to reports on the kinetics of IL-36R, IL-1RAcP, and IL-36 ligands, IL-36Ra exhibits the same binding activity to IL-36R and the IL-36R / IL-1RAcP complex. However, the binding activity of IL-36 ligands to IL-36R is equal to or slightly weaker than that of IL-36Ra. However, for the IL-36R / IL-1RAcP complex, it binds with more than 10 times the strength of IL-36Ra (Non-Patent Literature 19). The correlation of IL-36R information in the disease was clarified through studies using genetically modified mice with IL-36Ra (also known as IL-1F5), IL-36α (also known as IL-1F6), IL-36R (also known as IL-1RL2 or IL-1Rrp2), and IL-1RAcP. Not only in pathological patterns, but also in the skin of psoriasis patients, there have been reports of increased expression of IL-36R, IL-36α, and IL-36Ra (Non-Patent Literature 20). Furthermore, reports have demonstrated that the absence of IL-36R in imiquimod mice, using IL-36R gene knockout mice, resulted in a more severe suppression of inflammation than IL-17 or IL-22, and it is presumed to form a cytokine loop together with IL-17 or IL-23 (Non-Patent Literature 21). As a target for psoriasis treatment, reports have shown that IL-17 or IL-23 has a high effect on TNFα, and it is also presumed that IL-36 is the driver (Non-Patent Literature 22).

[0010] Clinically, there are reports that pustular psoriasis (generalized type) (GPP), especially severe psoriasis, is associated with IL-36Ra deficiency. IL-36Ra functional deficiency plays an important role in the disease (Non-Patent Literature 23), leading to the concept of DITRA (Deficiency of Interleukin - Thirty-six Receptor Antagonist). Genetically, DITRA is not only caused by homozygous variants; there are reports that heterozygous variants can also cause the disease. Acute generalized exanthematous pustulosis caused by drugs or herpetic pustular eczema occurring during pregnancy are also systematized as DITRA (Non-Patent Literature 24).

[0011] The balance of IL-36 ligand expression in autoimmune diseases, primarily psoriasis, has been reported by several teams. However, there are few cases of elevated IL-36β in tissues or lesions originating from patients. On the other hand, there is a significant increase in IL-36α and IL-36γ mRNA and a tendency for decreased IL-36Ra or IL-38 mRNA levels (Non-Patent Literature 25). In recent years, anti-IL-36 receptor antibodies (hereinafter appropriately referred to as "anti-IL-36R antibodies") have shown clinical efficacy in severe pustular psoriasis (systemic type), with reports of improvement in all cases after a single dose (Non-Patent Literature 26). While it remains unclear which IL-36 is most important to inhibit, it is known that even without DITRA, anti-IL-36R antibodies have shown high therapeutic efficacy at GPP levels. This report clearly demonstrates the potential of anti-IL-36R antibodies as a pharmaceutical target. Remaining issues can be considered related to pharmaceutical development, such as dosage or treatment method design, risk management, safety, and expansion of indications. <The Association of IL-36R with Diseases Other Than Psoriasis>

[0012] Reports indicate that IL-36 ligands are not only elevated in psoriasis, but also in autoimmune diseases such as systemic lupus erythematosus (SLE) or inflammatory bowel disease (IBD) where effective treatments have not yet been established (Non-Patent Literature 27). In recent years, patents have disclosed that IL-36Ra KO (gene knockout) mice develop arthritis (Patent Literature 1), and reports indicate that IL-36α is particularly elevated in steroid-resistant psoriatic arthritis (Non-Patent Literature 28).

[0013] IL-36 receptor or IL-36R agonist ligand expression has also been confirmed in the lungs, and reports indicate that it induces activation of macrophages or fibroblasts and collagen deposition. In recent years, there have been reports of an association between IL-36γ-positive macrophages and fibrosis (Non-Patent Literature 29), suggesting that IL-36R inhibition may have an effect on fibrosis. Furthermore, there are reports that the elimination or inhibition of IL-36R signaling has also shown therapeutic effects in pathological animal models of the kidneys (Non-Patent Literature 30) or intestines (Non-Patent Literature 31).

[0014] After the filing date (December 27, 2019; hereinafter referred to as the "priority date") of the priority-based application (Japanese Patent Application 2019-239130), a crystalline structure of IL-36R (PDB ID: 6U6U) was reported. Furthermore, in Patent Documents 6 and 7, which were published after the priority date of this case, anti-IL-36R antibodies and anti-IL-36 ligand antibodies were reported, respectively. [Prior Art Documents] [Patent Documents]

[0015] [Patent Document 1] Japanese Patent Application Publication No. 2017-055680 [Patent Document 2] International Publication No. 2013 / 074569 [Patent Document 3] International Publication No. 2016 / 168542 [Patent Document 4] International Publication No. 2013 / 180238 [Patent Document 5] International Publication No. 2006 / 106366 [Patent Document 6] International Publication No. 2020 / 018503 [Patent Document 7] International Publication No. 2020 / 065594 [Non-Patent Documents]

[0016] [Non-patent literature 1] J. Allergy Clin. Immunol., 2016, 138[4]:984-1010 [Non-patent literature 2] J. Biol. Chem., (2004), 279

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[12] : e114604. [Summary of the Invention]

[0017] [Problem to be solved by the invention] <Function of each IL-36R agonist ligand>

[0018] For pharmaceuticals used in treatment, not only efficacy but also high safety is required. In addition to the infections reported to date, the risk of new infections or cancer progression must also be considered. This safety requirement includes not only a short-term perspective but also a long-term perspective following clinical trials. However, antibody pharmaceuticals targeting cytokines, such as anti-TNF antibodies, widely used in the treatment of rheumatism or psoriasis, have shown high therapeutic efficacy that low-molecular-weight pharmaceuticals have not been able to achieve to date. On the other hand, side effects, as revealed by clinical trials, are not easily predicted. Therefore, if equivalent efficacy can be obtained, drugs with lower risks are naturally advantageous.

[0019] IL-36α, IL-36β, and IL-36γ, like IL-17A, exhibit an effect of increasing the expression of antimicrobial peptides, with IL-36β showing the strongest effect (Non-Patent Literature 4). Furthermore, since the mRNA of IL-36β in mononuclear cells such as dendritic cells or macrophages shows little change in response to LPS stimulation (Non-Patent Literature 25), it is believed that IL-36β plays a different role in immune maintenance than IL-36α and IL-36γ. There are reports of IL-36β expression in glial cells in rodents (Non-Patent Literature 32) or its importance in the effector function of T cells (Non-Patent Literature 33). The increased mortality rate from herpesvirus infection in mice with only the IL-36β gene knockout (Non-Patent Literature 34) may not be accidental.

[0020] In autoimmune diseases, primarily psoriasis, there are almost no reports of significant increases in IL-36β. On the other hand, there are reports of high concentrations of IL-36β in the blood of healthy individuals (Non-Patent Literature 35). IL-36β and γ are closely related to host defense mechanisms such as T cell survival and IFNγ expression (Non-Patent Literature 36), leading to an increase in MHC Class II and regulating the expression of novel cancer-targeting molecules such as CD40 (Non-Patent Literature 9). Therefore, the pathophysiological significance of IL-36β remains unclear. If anti-IL-36R antibodies, as with JAK inhibitors, show undeniable contributions to cancer beyond shingles or other infections, further drug development becomes difficult. Therefore, selective IL-36 inhibitory antibodies could be developed. Thus, considering the therapeutic benefits and side effects, there is a desire to specifically inhibit IL-36α and IL-36γ, but currently, no such drugs exist. <Antibody that inhibits receptor function>

[0021] Generally speaking, when a cytokine receptor has multiple ligands, it is difficult to selectively block ligand signaling by using antibodies that bind to the receptor. As described in non-patent literature, anti-IL-1 receptor antibody systems block the signaling of both IL-1α and IL-1β (Non-patent Literature 37), and anti-IL-17RA and anti-IL-17RC antibody systems block the signaling of both IL-17A and IL-17F, respectively (Non-patent Literature 38). There is also no record of IL-36 signaling-specific blocking antibodies in patent literature (Patent Literature 2, Patent Literature 3). While the blocking morphology of the antibodies reported in these cases is not clearly defined, if the antibody blocks the formation of a receptor-ligand complex, considering the molecular size of the cytokines or their receptors and the antibody, since the antibody molecular size is larger than the site where the IL-36 ligand binds to the IL-36 receptor, selectively blocking each IL-36 ligand is considered difficult. It is generally believed that an anti-receptor antibody system that selectively blocks only specific ligands does not exist. [Means used to solve the problem]

[0022] As a result of repeated and in-depth research, the inventors discovered a novel method for obtaining a safe and effective inhibitor for efficient treatment by binding to IL-36R without blocking IL-36β, but only blocking IL-36α and IL-36γ, which have been reported to be elevated in autoimmune diseases and related to pathology.

[0023] That is, the purpose of the present invention is to provide an IL-36R neutralizing antibody that specifically binds to human IL-36R and blocks the message of the IL-36R causative agent. It is an IL-36R message-specific blocking antibody or its fragment, or a derivative thereof, in which the message blocking of IL-36β among IL-36α, β, and γ is weak or non-blocking.

[0024] Furthermore, the purpose of this invention is to inhibit the abnormal activation of the immune system mediated by IL-36R, and to provide a means of treating autoimmune diseases or cancer.

[0025] That is, the present invention relates to the following. [Item 1] An anti-human IL-36R antibody or a fragment thereof, or a derivative thereof, which is an antibody or a fragment thereof, or a derivative thereof, targeting human IL-36R, wherein, among the three human IL-36R agonist ligands IL-36α, β and γ, the 50% blocking concentration (IC50) for the signal of at least one ligand is more than 10 times the 50% blocking concentration (IC50) for the signal of at least one other ligand. [Item 2] The anti-human IL-36R antibody or a fragment thereof, or a derivative thereof, as stated in Item 1, wherein, in a Cell ELISA using IL-36R-expressing cells, it shows a 50% effective concentration (EC50) of less than 1 × 10⁻⁷ M. [Item 3] The anti-human IL-36R antibody or fragment thereof, or derivative thereof, as described in Item 1 or 2, wherein the 50% blocking concentration for the signaling of the human IL-36R ligand IL-36β is more than 10 times the 50% blocking concentration for the signaling of the human IL-36R ligands IL-36α and γ. [Item 4] The anti-human IL-36R antibody or fragment thereof, or derivative thereof, as described in Item 1 or 2, wherein the blocking of the signaling of the human IL-36R ligands IL-36α and γ is more than 50%, and the blocking of the signaling of IL-36β is less than 50%. [Item 5] The anti-human IL-36R antibody or fragment thereof, or derivative thereof, as described in Item 3 or 4, wherein at a concentration of 0.2 ng / mL or higher, the 50% blocking concentration for the signaling of IL-36β is more than 10 × 10⁻⁸ M. [Item 6] The anti-human IL-36R antibody or fragment thereof, or derivative thereof, as described in any of items 1 to 5, binds to domain I of human IL-36R. [Item 7] The anti-human IL-36R antibody or fragment thereof, or derivative thereof, as described in any of items 1 to 6, binds to at least two regions of the amino acid sequence (Sequence Number 11) of human IL-36R, including the region consisting of amino acid residues 1 to 8 from the N-terminus, the region consisting of amino acid residues 25 to 32, and the region consisting of amino acid residues 81 to 88. [Item 8] The anti-human IL-36R antibody or fragment thereof, or derivative thereof, as described in any of items 1 to 7, binds to the 4th lysine residue and the 28th isoleucine residue of the amino acid sequence (Sequence Number 11) of human IL-36R. [Item 9] The anti-human IL-36R antibody or fragment thereof, or derivative thereof, as described in any of items 1 to 8, comprises at least a heavy chain variable sequence, and the aforementioned heavy chain variable sequence (1) comprises an amino acid sequence of sequence number 23 or 25 (XH001 or XH002) as a CDR-H1 sequence.(1b) an amino acid sequence having at least 80% similarity to the amino acid sequence of (1a) above, or (1c) an amino acid sequence in which one or two amino acid residues of the amino acid sequence of (1a) above are substituted, deleted or added; (2) an amino acid sequence comprising (2a) selected from sequence numbers 27, 29, 31 and 213 (XH003~XH005 and XH007) as a CDR-H2 sequence, (2b) an amino acid sequence having at least 88% similarity to the amino acid sequence of (2a) above, or (2c) an amino acid sequence in which one or two amino acid residues of the amino acid sequence of (2a) above are substituted, deleted or added; (3) An amino acid sequence comprising (3a) any one of sequence numbers 33, 215 and 217 (XH006, XH008 and XH009) as a CDR-H3 sequence, (3b) an amino acid sequence having at least 83% similarity to the amino acid sequence of (3a) above, or (3c) an amino acid sequence in which one or two amino acid residues of the amino acid sequence of (3a) are substituted, deleted or added. [Item 10] An anti-human IL-36R antibody or a fragment thereof, or a derivative thereof, as described in Item 9, wherein the frame sequence of the aforementioned heavy chain variable sequence contains frame sequences of various types of immunoglobulins of humans or non-human animals including monkeys, mice or rats. [Item 11] The anti-human IL-36R antibody or fragment thereof, or derivatives thereof, as described in Item 9 or 10, wherein the aforementioned heavy chain variable sequence comprises (a) an amino acid sequence selected from sequence number 45 (Pro000713v), sequence number 47 (Pro000722v), sequence number 49 (Pro001558v), sequence number 51 (Pro001562v), sequence number 53 (Pro001554v), sequence number 55 (Pro001566v), sequence number 57 (Pro001570v), sequence number 59 (Pro001574v), sequence number 177 (Pro002817v), sequence number 181 (Pro002818v), and sequence number 185 (Pro002819v), (b) an amino acid sequence having at least 80% similarity to the amino acid sequence of (a) above, or (c) an amino acid sequence in which 1 to 12 amino acid residues of the amino acid sequence of (a) above are substituted, deleted, or added. [Item 12] An anti-human IL-36R antibody or fragment thereof, or a derivative thereof, as described in any of items 1 to 11, comprising at least a light chain variable sequence, wherein the light chain variable sequence (1) comprises an amino acid sequence of (1a) selected from sequence numbers 35, 37, and 219 (XL001, XL002, and XL006) as a CDR-L1 sequence,(1b) An amino acid sequence having at least 84% similarity to the amino acid sequence of (1a) above, or (1c) An amino acid sequence in which one or two amino acid residues of the amino acid sequence of (1a) above are substituted, deleted, or added; (2) An amino acid sequence comprising (2a) selected from sequence numbers 39, 41, and 221 (XL003, XL004, and XL007) as a CDR-L2 sequence, (2b) an amino acid sequence having at least 85% similarity to the amino acid sequence of (2a) above, or (2c) an amino acid sequence in which one or two amino acid residues of the amino acid sequence of (2a) above are substituted, deleted, or added; (3) An amino acid sequence comprising (3a) selected from sequence numbers 43, 223, and 225 (XL005, XL008, and XL009) as a CDR-L3 sequence, (3b) An amino acid sequence having at least 88% similarity to the amino acid sequence of (3a) above, or (3c) An amino acid sequence in which one or two amino acid residues of the amino acid sequence of (3a) are substituted, deleted, or added. [Item 13] The anti-human IL-36R antibody or fragment thereof, or derivatives thereof, as described in Item 12, wherein the aforementioned light chain variable sequence further contains various types of framework sequences of human or non-human animal immunoglobulins including monkeys, mice, or rats. [Item 14] The anti-human IL-36R antibody or fragment thereof, or derivative thereof, as described in Item 12 or 13, wherein the aforementioned light chain variable sequence comprises (a) an amino acid sequence selected from sequence number 61 (Pro000714v), sequence number 63 (Pro001332v), sequence number 65 (Pro001333v), sequence number 67 (Pro001334v), sequence number 179 (Pro002821v), sequence number 183 (Pro002823v), and sequence number 187 (Pro002822v), (b) an amino acid sequence having at least 80% similarity to the amino acid sequence of (a) above, or (c) an amino acid sequence in which 1 to 10 amino acid residues of the amino acid sequence of (a) above are substituted, deleted, or added. [Item 15] An anti-human IL-36R antibody or fragment thereof, or a derivative thereof, as described in any of items 1 to 14, wherein the constant heavy chain region and / or constant light chain region are constant regions of various types of immunoglobulins of humans or non-human animals including mice, rats, or monkeys. [Item 16] An anti-human IL-36R antibody or fragment thereof, or a derivative thereof, as described in any of items 1 to 15, which is a Fab, scFv, Diabody, or bispecific antibody, or a derivative thereof. [Item 17] A nucleic acid molecule comprising a polynucleotide sequence encoding an anti-human IL-36R antibody or fragment thereof, or a derivative thereof, as described in any of items 1 to 16.[Item 18] A selection vector or expression vector comprising at least one nucleic acid molecule as described in Item 17. [Item 19] A recombinant cell having a vector as described in Item 18 introduced therein. [Item 20] A method for producing an anti-human IL-36R antibody or a fragment thereof, or a derivative thereof, as described in any of Items 1-16, comprising culturing a recombinant cell as described in Item 19. [Item 21] A pharmaceutical composition comprising one or more of a group of an anti-human IL-36R antibody or a fragment thereof, or a derivative thereof, as described in any of Items 1-16, a nucleic acid molecule as described in Item 17, a vector as described in Item 18, and a recombinant cell as described in Item 19. [Item 22] A pharmaceutical composition as described in Item 21, administered to humans to modulate or modify an immune response. [Item 23] A pharmaceutical composition as described in Item 21 or 22, administered to humans at a frequency of less than once per week. [Item 24] A pharmaceutical composition of any of items 21-23 is used for the treatment and / or prevention of cancer or autoimmune diseases in vertebrates. [Item 25] A pharmaceutical composition of any of items 21-24 further contains pharmaceutically permissible diluents, drug carriers, and / or other additives. [Item 26] A pharmaceutical composition of any of items 21-25 further contains a second active ingredient. [Item 27]As in the pharmaceutical composition of item 26, the aforementioned second active ingredient is selected from azelastine, oxatomide, mequitazine, fexofenadine, epinastine, ebastine, cetirizine, levocetirizine, bepotastine, emedastine, olopatadine, loratadine, levocabastine, ozagrel, seratrodast, ramatroban, pranlukast, montelukast, zafirlukast, subplatast, and diphenhydramine. One or more of the group consisting of dimenhydrinate, diphenylpyraline, clemastine, chlorpheniramine, triprolidine, promethazine, alimethazine, hydroxyzine, homochlorcyclizine, cyproheptadine, mesalazine, interferon beta-1b, interferon beta-1a, fingolimod hydrochloride, natalizumab, glatiramer acetate, dimethyl fumarate, etretinate, tacrolimus, mercaptopurine, azathioprine, and apremilast. [Item 28]For pharmaceutical compositions such as those listed in items 26 or 27, the aforementioned second active ingredient is selected from corticosteroids, antiemetics, ondansetron hydrochloride, granisetron hydrochloride, metroclopramide, domperidone, haloperidol, cyclizine, lorazepam, prochlorperazine, dexamethasone, levomepromazine, tropisetron, cancer vaccines, GM-CSF inhibitors, GM-CSF, DNA vaccines, cell-based vaccines, dendritic cell vaccines, recombinant viral vaccines, heat shock protein (HSP) vaccines, homogeneous tumor vaccines, autologous tumor vaccines, analgesics, ibuprofen, and naproxen. Trisalicylic acid choline magnesium, oxycodone hydrochloride, anti-angiogenic drugs, anti-angiogenic drugs, anti-PD-1 antibodies, nivolumab, pembrolizumab, tislelizumab, anti-PD-L1 antibodies, atezolizumab, avelumab, durvalumab, anti-CTLA4 antibodies, ipilimumab, anti-CD20 antibodies, rituximab, anti-HER2 antibodies, trastuzumab, anti-CCR4 antibodies, mogamulizumab, anti-VEGF antibodies, bevacizumab, anti-VEGF receptor antibodies, soluble VEGF receptor fragments, anti-TWEAK antibodies, anti-TWEAK receptor antibodies, soluble TWEAK receptor fragments, AMG 706, AMG386, Anti-α4β7 antibody, Bevacizumab, Etrolizumab, Anti-SIRPα antibody, Anti-CD40 antibody, Anti-CD40L antibody, Anti-proliferative drugs, Farnesyl protein transferase inhibitors, αvβ3 inhibitors, αvβ5 inhibitors, p53 inhibitors, Kit receptor inhibitors, ret receptor inhibitors, PDGFR inhibitors, Growth hormone secretion inhibitors, Angiopoietin inhibitors, Tumor-infiltrating macrophage inhibitors, c-fms The invention comprises one or more of the following: an inhibitor, an anti-c-fms antibody, a CSF-1 inhibitor, an anti-CSF-1 antibody, a soluble c-fms fragment, pegvisomant, gemcitabine, panitumumab, irinotecan, a TNFα inhibitor, an IL-17A inhibitor, an IL-17F inhibitor, an IL-17RA inhibitor, an IL-23p19 inhibitor, an IL-23p40 inhibitor, and an IL-1RAcP inhibitor, and a group consisting of SN-38. [Effects of the Invention]

[0026] The IL-36R message-specific blocking antibody or its fragment or derivative thereof of the present invention, by specifically binding to human IL-36R, blocks specific messages in the IL-36R message and regulates cellular responses, thereby having therapeutic effects on autoimmune diseases or cancer.

Implementation Method

[0027] Hereinafter, the present invention will be described based on specific embodiments, but the present invention is not limited to these embodiments in any way. Furthermore, all patent gazettes, patent application publications, and non-patent gazettes cited in this specification are incorporated herein by reference in their entirety for all purposes. [IL-36]

[0028] In this invention, "IL-36" refers to the ligand of interleukin 36, one of the interleukins. That is, "IL-36" includes three ligands: IL-36α, IL-36β, and IL-36γ. The amino acid sequences of human IL-36α, IL-36β, and IL-36γ proteins are shown in sequence numbers 1, 3, and 5, respectively, and the corresponding base sequences of human IL-36α, IL-36β, and IL-36γ genes are shown in sequence numbers 2, 4, and 6, respectively. Furthermore, Figure 1 shows the sequence alignment comparing the amino acid sequences of the active forms of human IL-36α, β, and γ. When describing a specific ligand among these three IL-36R ligands, α, β, or γ is appended after "IL-36" to specify which ligand is being referred to.

[0029] Similar to the IL-1 receptor to which IL-1 binds, the IL-36 receptor (IL-36R) to which IL-36 binds has a natural antagonist ligand that inhibits the binding of IL-36 (IL-36R antagonist ligand: hereinafter also referred to simply as "IL-36R antagonist"). Known natural IL-36R antagonists are IL-36Ra and IL-38. The amino acid sequences of the human IL-36Ra and IL-38 proteins are shown in sequence numbers 7 and 9, respectively, and the corresponding base sequences of the human IL-36Ra and IL-38 genes are shown in sequence numbers 8 and 10, respectively.

[0030] The genes for IL-36 and IL-36R antagonists are located at the same locus and are known to function as important physiologically active substances related to host defense in many species. IL-36 and IL-36R antagonists are primarily found in the skin, and secondarily in mucosal and immune tissues. Furthermore, in autoimmune diseases, elevated mRNA levels of IL-36 and IL-36R antagonists have been reported in various cell types, including keratinocytes, fibroblasts, epithelial cells, leukocytes (primarily T cells), dendritic cells, and macrophages. Both IL-36 and IL-36R antagonists exist in inactive and active forms. The conversion of the inactive form to the active form primarily requires digestion by proteases (digestive enzymes) produced by immune-active cells; the digestive enzymes and the sequences to be digested vary depending on the ligand. Unlike IL-36, the IL-36R antagonist IL-36Ra becomes active simply by removing the N-terminal methionine. The active form of another IL-36R antagonist, IL-38, is not yet clear.

[0031] Unless otherwise specified, the terms "IL-36" and "IL-36R antagonist" in this specification refer to the active form. The IL-36 ligand prior to protease digestion is described as "IL-36 precursor" or "IL-36α precursor," with the addition of "precursor." [IL-36R and IL-36R complex]

[0032] In this invention, "IL-36R" refers to the receptor to which IL-36 binds, i.e., the IL-36 receptor. Figure 2 shows the amino acid sequence of human IL-36R. Figure 3 schematically shows the three-dimensional structure of human IL-36R. The amino acid sequence of the human IL-36R protein can be referenced by NCBI accession number NP_001338375.1 or Uniprot accession number Q9HB29, etc.

[0033] Furthermore, multiple isoforms of human IL-36R have been reported. Unless otherwise specified, when referred to as "human IL-36R" in this specification, it refers to isoform "a", and further refers to a protein whose signal peptide has been cleaved through post-translational modification (i.e., a protein with the amino acid sequence shown in sequence number 11 (Pro001391)).

[0034] It is known that many vertebrates possess IL-36 ligands and IL-36R proteins. The amino acid sequences of IL-36R are known in humans (sequence number 13: Q9HB29|ILRL2_HUMAN), cynomolgus monkeys (sequence number 15: A0A2K5WSK9_MACFA), rhesus monkeys (sequence number 17: A0A1D5RBJ8_MACMU), mice (sequence number 19: Q9ERS7|ILRL2_MOUSE), and rats (sequence number 21: Q62929|ILRL2_RAT). The amino acid sequences of IL-36R in other animal species can also be found in databases such as NCBI or EMBL. Figure 4 shows a sequence alignment comparison of the amino acid sequences of IL-36R in humans, cynomolgus monkeys, rhesus monkeys, mice, and rats, which contain the signaling peptide.

[0035] IL-36R forms a complex with IL-1RAcP, a signal transduction receptor common to the IL-36 and IL-1 receptor families (Non-Patent Document 2). This complex is hereinafter referred to as the "IL-36R complex". [IL-36R antagonist and IL-36R neutralizing antibody]

[0036] In this invention, "IL-36R antagonist ligand" or "IL-36R antagonist" refers to all ligands that can bind to IL-36R and block IL-36R signaling. Naturally occurring IL-36 receptor antagonists are labeled "IL-36Ra". The forms of IL-36R antagonists can include high-molecular-weight antibodies, variant antibodies, peptides, aptamers, etc., or various low-molecular-weight molecules. Furthermore, even molecules that have not been reported to date, any molecule that can bind to IL-36R and block its signaling, are also included in IL-36R antagonists. Among anti-IL-36R antibodies that can bind to IL-36R, IL-36R antagonists that can block the signaling of IL-36R agonist ligands are described herein as "IL-36R neutralizing antibodies". [PAN antibodies and IL-36R signaling-specific blocking antibodies]

[0037] Unless otherwise specified, the aforementioned IL-36R neutralizing antibodies are those that block all messages from various IL-36R agonist ligands of IL-36α, β, and γ. The IL-36R neutralizing antibodies described in this invention are also sometimes referred to as "PAN antibodies" by adding the Greek prefix meaning "all". Antibodies that block one or more messages from various IL-36α, β, and γ ligands, or antibodies that block one or more messages while not blocking one or more messages, or whose 50% blocking concentration (IC50) of one or more messages is more than 10 times greater than the 50% blocking concentration (IC50) of other messages, are described as "IL-36R message-specific blocking antibodies". Antibodies with an IC50 10 times or more greater mean that their blocking activity is 10 times or more weaker. Unless otherwise specified, in this invention, "IL-36R signaling-specific blocking antibody" refers to an antibody that, compared to IL-36α and IL-36γ, has a relatively weaker signaling blocking effect on IL-36β, or does not block IL-36β signaling at all. [Definition of Antibody]

[0038] An antibody system refers to a glycoprotein containing at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. The heavy chain includes a variable region (VH) and a constant region, the constant region containing three domains: CH1, CH2, and CH3. The light chain includes a variable region (VL) and a constant region. The constant region contains one domain: CL. The constant region of the light chain exists in two forms: a λ chain and a κ chain. The constant region of the heavy chain contains γ, μ, α, δ, and ε chains, and depending on the heavy chain, isotypes of antibodies called IgG, IgM, IgA, IgD, and IgE exist. The VH and VL regions are further subdivided into four more preserved regions (FR-1, FR-2, FR-3, FR-4) called framework regions (FR), and three variable regions (CDR-1, CDR-2, CDR-3) called complementarity-determining regions (CDR). The VH region, from the amino terminus to the carboxyl terminus, contains three CDRs and four FRs arranged in the order FR-1, CDR-1 (CDR-H1), FR-2, CDR-2 (CDR-H2), FR-3, CDR-3 (CDR-H3), FR-4. The VL region, from the amino terminus to the carboxyl terminus, contains three CDRs and four FRs arranged in the order FR-1, CDR-1 (CDR-L1), FR-2, CDR-2 (CDR-L2), FR-3, CDR-3 (CDR-L3), FR-4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen.

[0039] The antibody of the present invention, as long as it has the IL-36R signal-specific blocking activity described later, can be an antibody fragment (e.g., an antigen-binding fragment) and / or a derivative. Examples of antibody fragments include F(ab')2, Fab, Fv, etc. Examples of antibody derivatives include antibodies that artificially introduce amino acid variations into a constant region, antibodies that alter the structural domain composition of a constant region, antibodies with two or more Fc morphologies per molecule, antibodies composed only of heavy or light chains, glycan variant antibodies, bispecific antibodies, antibodies or antibody fragment compounds, or antibody conjugates that bind to proteins other than antibodies, antibody enzymes, nano-antibodies, tandem scFvs, bispecific tandem scFvs, micro-diabody antibodies, VHH, etc. Furthermore, when referred to simply as "antibody" in the present invention, unless otherwise explicitly stated, it also includes antibody fragments and / or derivatives.

[0040] Furthermore, monoclonal antibodies, classically speaking, refer to antibody molecules derived from a strain of cells producing a single antibody, but specifically refer to a single type of antibody molecule containing a combination of VH and VL composed of specific amino acid sequences. Monoclonal antibodies can also be obtained from nucleic acid molecules containing gene sequences of amino acids encoding proteins of their antibodies, and antibodies can be genetically engineered using such nucleic acid molecules. Furthermore, techniques well known to those skilled in the art include using genetic information such as H chains, L chains, variable regions, or CDR sequences to modify antibody binding or specificity, or modifying antibodies from animals such as mice into human-type antibodies to create antibodies suitable for therapeutic formulations. Additionally, human monoclonal antibodies can be obtained by using genetically modified animals with introduced human antibody genes as animals to sensitize antigens. In addition, as a method that does not require sensitization of animals, a phage gene library (human antibody phage display) expressing the antigen-binding region of human antibodies or a portion thereof is used to obtain phage strains composed of antibodies that specifically bind to the corresponding antigen or specific amino acid sequences. The technique for producing human antibodies from this information is also suitable for those skilled in the art (e.g., see the review by Keio J. Med., (2011), 60:37-46). Furthermore, when designing antibodies for administration to animals other than humans, similar to the humanization technique, those skilled in the art can appropriately use CDR or variable region amino acid sequence information for design.

[0041] The specificity of an antibody refers to the high antigen-antibody reaction exhibited by the antibody against a particular antigen. The determination of the antigen-antibody reaction can be performed by a person skilled in the art, appropriately selecting a solid-phase or liquid-phase system for the binding assay. Examples of such methods include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), surface plasmon resonance (SPR), fluorescence resonance energy transfer (FRET), and luminescence resonance energy transfer (LRET). Furthermore, when determining such antigen-antibody binding, the antibody and / or antigen can be labeled with enzymes, fluorescent substances, luminescent substances, radioactive isotopes, etc., and the antigen-antibody reaction can be detected using a assay method suitable for the physical and / or chemical properties of the labeled substance. [IL-36R Message and Screening Methods for Antibodies Inhibited by IL-36R Message]

[0042] IL-36 forms the IL-36R complex with IL-36R and IL-1RAcP, conducting signal transduction mediated by the TIR domain. The signal transduction associated with MyD88 has been studied in detail for TLR (Toll-Like Receptor) or IL-1R (Interleukin-1 Receptor) signals, and can be evaluated by NFkB phosphorylation or by reporter gene analysis mediated by the IL-8 promoter. Furthermore, the cell activation resulting from IL-36 signaling can also be evaluated by assessing the increased expression of mRNA or the produced protein itself. In this invention, to enable the screening of antibodies targeting signal transduction, a novel analysis system using the NFkB reporter gene has been constructed based on publicly available information. This evaluation system is used to detect IL-36R neutralizing antibodies and IL-36R signal-specific blocking antibodies. As shown in Example 6, the inventors, through screening using binding assays and reporter gene assays based on the aforementioned IL-36α information, extracted 111 antibodies that inhibited IL-36α to form fusion tumors. After evaluating the inhibitory activity of IL-36β and IL-36γ in these 111 antibody fusion tumors, the inhibition of IL-36α and IL-36γ information showed an almost 1:1 relationship, meaning the same degree of inhibition, but multiple antibodies inhibiting IL-36α were obtained while not inhibiting IL-36β. After purifying these antibody fusion tumors using a limiting dilution method (single-strain), the resulting antibodies almost completely inhibited the information of IL-36α and IL-36γ, but acted as partial antagonists for IL-36β information. Based on the analysis of screening for binding to IL-36R and reporter gene analysis based on IL-36α, it was determined that screening based on reporter gene analysis of IL-36α and IL-36β was the best method. After further screening, antibodies were obtained that did not block IL-36β relative to the blocking of IL-36α, and almost completely blocked the signals of IL-36α and IL-36γ.

[0043] In the screening systems described in Examples 6 and 7 below, the concentration of the IL-36R neutralizing antibody cannot be adjusted. Furthermore, since a deviation of 1 point is considered large, the screening system defines a reduction of more than 40% in information as "inhibiting activity". In cases where inhibition cannot be detected, there is a particular tendency for the deviation to increase, therefore no threshold is set for the upper limit of information. The screening using the culture supernatant of fusion tumors is performed with a ligand concentration of 150 ng / mL as the final concentration. However, in the test to evaluate the inhibitory strength of the purified antibody, the dynamic range or deviation is considered, and an appropriate concentration is set for each ligand in each test. As described in Examples 6 and 7 below, the antibody of the present invention differs from the conventional anti-IL-36R antibodies described in the aforementioned patent and non-patent documents in that it inhibits IL-36α and IL-36γ but not IL-36β, or has a relatively weak inhibitory strength. In Examples 13, 14 and 17 described later, a 50% blocking concentration (IC50) was confirmed using purified recombinant antibodies.

[0044] This case is the first to report antibodies that do not block the signaling caused by a specific ligand, not only against IL-36 but also against interleukin receptors bound by multiple ligands. When referring to the method described in this case, antibodies that do not block IL-36α or antibodies that block IL-36γ can be detected by screening.

[0045] In previous clinical trials of the anti-IL-36R neutralizing antibody MAB92 (Spesolimab) in patients with severe pustular psoriasis (generalized type), no IL-36Ra deficiency was observed, and a single dose showed therapeutic efficacy (Non-Patent Literature 26). Pustular psoriasis (generalized type) is generally considered a different disease from psoriasis vulgaris. However, anti-IL-36R neutralizing antibodies have also shown therapeutic efficacy in patients with pustular psoriasis (generalized type) who are not equivalent to patients with IL-36Ra deficiency. Reports of changes in psoriasis-related cytokines such as IL-17 or IL-23, or further, based on the non-patent literature described below, suggest that anti-IL-36R systems are effective in psoriasis-related diseases. This implies the possibility that anti-IL-36R neutralizing antibodies may be effective not only in pustular psoriasis (generalized type) but also in psoriasis vulgaris. Here, when examining the contribution of IL-36 ligands to psoriasis, it has been reported that IL-36γ is correlated with the PASI score, a major assessment item for psoriasis (J. Invest. Dermatol., (2015), 135[4]: 1025-32). It is believed that in about 30% of psoriatic arthritis cases, there is an increase in IL-36α (Non-Patent Literature 28), indicating that the inhibitory relationship between IL-36α and γ is extremely important. On the other hand, the extent to which IL-36β contributes to autoimmune diseases, primarily psoriasis, is still unknown. Compared to the concentration in tissues such as skin, the concentration of IL-36β in the blood is relatively high, and it has been reported that it has the strongest effect or binding system among IL-36, especially with a high ability to produce antimicrobial peptides (Non-Patent Literature 4). Therefore, the communication of all ligands of IL-36 may not achieve the desired effect in diseases where the host defense mechanism, primarily due to infection, is compromised, and viral or bacterial infections further exacerbate the condition. IL-18, belonging to the same IL-1 family as IL-36, has been reported to be activated by Staphylococcus aureus enzymes. Therefore, it is not surprising that IL-36, particularly when constantly activated in the intestinal environment or palms and soles, contributes to the host's defense mechanisms. However, the role of IL-36 in the host's defense mechanisms remains unclear.

[0046] In summary, the IL-36R antagonist of the present invention, specifically, is an IL-36R signal-specific blocking antibody, which has the potential to become a therapeutic or preventive drug for various diseases associated with IL-36R signals, and has the potential to overcome the side effects such as infections caused by completely blocking IL-36R signals and the pathological deterioration of the diseases caused by them, and obtain a stable therapeutic effect.

[0047] The importance of IL-36 was recognized through reports of IL-36Ra dysfunction in patients with familial gene mutations that cause severe pustular psoriasis (generalized type) (Non-Patent Literature 23). Compared to normal IL-36Ra, the IC50 of IL-36R signaling in this dysfunctional IL-36Ra is about 10 times larger. Therefore, clinically, an IC50 of less than 10 × 10⁻⁸ M is considered a significant signaling block, and the usefulness of antibodies is judged based on IC50. Therefore, in this invention, an antibody that does not block IL-36β means an antibody with a 50% blocking concentration (IC50) of 10 × 10⁻⁸ M or higher for IL-36β-induced signals, and an IL-36R signal-specific blocking antibody means an antibody with a 50% blocking concentration (IC50) of 10 × 10⁻⁸ M or lower for IL-36α and IL-36γ-induced signals, and that does not block IL-36β-induced signals, or an antibody whose blocking activity for IL-36β-induced signals is lower than that for IL-36α and IL-36γ-induced signals (preferably, the IC50 of IL-36β-induced signals is more than 10 times that of IL-36α and IL-36γ-induced signals). Furthermore, the IL-36R signaling-specific blocking antibody of the present invention preferably blocks more than 50%, particularly more than 60%, and further more than 70% of the signaling of human IL-36R ligands IL-36α and γ, and only blocks less than 50%, particularly less than 40%, and further less than 30% of the signaling of human IL-36R ligand IL-36β. As shown in Example 13 below, the antibody and recombinant antibody (recombinant antibody) derived from fusion tumors obtained by the inventors block the signaling caused by IL-36α and IL-36γ, but do not block the signaling caused by IL-36β. Furthermore, as shown in Example 14 below, even if the concentration of the ligand is reduced or the antibody concentration is increased, IL-36β is not blocked. Even at antibody concentrations equivalent to the trough value of anti-IL-4 receptor antibody or anti-IL-17 receptor antibody, the antibody does not block IL-36β, exhibiting the characteristic of not showing concentration-dependent blocking.

[0048] As explained above, the antibodies obtained in the following examples are antibodies selected based on the signal blocking strength of IL-36α, IL-36β, and IL-36γ. They are the first antibodies to show selective signal blocking of IL-36R and the first antibodies to show no blocking of IL-36β.

[0049] However, the antibodies of the present invention are not limited to the specific antibodies obtained in the examples described below. Those skilled in the art can obtain novel IL-36R message-specific blocking antibodies with different amino acid sequences or antigenic determinants from the antibodies obtained in the examples by appropriately referring to conventional techniques and using the various information or techniques detailed in this specification, without excessive trial and error. For example, other IL-36R message-specific blocking antibodies can be obtained by using a screening method that compares the message-blocking strength of IL-36α, IL-36β, and IL-36γ detailed in this specification. Furthermore, IL-36R message-specific blocking antibodies can be obtained efficiently by referring to the information on antigenic determinants described below. Furthermore, other IL-36R message-specific blocking antibodies can be further obtained by detecting antibodies that compete with antibodies actually obtained in this manner using the previously known methods described below. In other words, antibodies with the same function can also be obtained by referring to the information described in this specification for immunization. Antibodies obtained in this manner, as long as they correspond to the definition of antibodies in this invention, are also included in the antibodies of this invention. [Antigen determinant site]

[0050] Unexpectedly, the binding site of the IL-36R antagonist antibody was not known in the prior art described in the patent literature. In one non-patent literature (MAbs., (2017), 9[7]: 1143-54), the antigenic determinant of MAB92 was reported as a development candidate. However, the antigenic determinant of the antibody was evaluated by the deuterium substitution method. According to another non-patent literature (MAbs., (2018), 10[2]: 204-9), the method of obtaining the antigenic determinant showed an unclear possibility. In fact, the area obtained as the antigenic determinant of MAB92 by this method is considered to be a physically impossible area to function as an antigenic determinant, considering the size of the binding surface formed by the Fab and CDR of the antibody. Therefore, although several antibodies have been reported as IL-36R antagonists, it is unclear whether the antagonist system of the prior art inhibits the binding of IL-36R to the IL-36R agonist ligand or inhibits the formation of the IL-36R-IL-1RAcP complex.

[0051] Therefore, the inventors evaluated the binding of the antibody to IL-36R in Example 9 described below and confirmed that the IL-36R message-specific blocking antibody discovered in this invention binds to human IL-36R but not to mouse IL-36R (data for mouse IL-36R is not shown). Furthermore, in Example 10 described below, the extracellular domain necessary for message-specific blocking was determined. The extracellular domain of IL-36R has 316 residues and consists of three Ig-like domains. Regarding the definition of the domains of IL-36R, since the structures reported in the literature are presumptive structures, there are various interpretations. However, unless otherwise specified in this specification, the sequence alignment shown in Figure 4 is followed, with amino acids 1-100 in sequence number 11 (Pro001391) designated as domain I, amino acids 101-198 as domain II, and amino acids 199-316 as domain III. For each domain, six chimeric receptors derived from human or mouse domains were designed, selected, and combined, and their binding was evaluated after gene delivery into cells.

[0052] In Example 11 described later, the antigenic determinants of the antibody determined in Example 10 were prepared by replacing all of the amino acid sequences of domain I and part of domain II with mouse-type receptors. The results of crystallographic analysis of the antigenic determinants of the antibody were reported to be that the antigenic determinants of continuous amino acid sequences were mostly from 5 to 8 residues (J. Immunol., (2013), 191[3]: 1428-35). Therefore, receptors were designed by replacing the human sequence with the mouse sequence by 8 residues starting from the first amino acid from the N-terminus, and by replacing the human sequence with the mouse sequence by 8 residues starting from the fifth amino acid, in a way that the antigenic determinants of 5 to 8 residues could be identified. Specifically, the receptor was made to grasp the antigenic determinants of the IL-36R message that specifically inhibit the antibody during transient expression of the cell. As a result, as shown in Example 11 below, it was found that the IL-36R message-specific blocking antibody provided by the present invention is an antibody that binds to domain I of IL-36R. Furthermore, by substituting three regions in domain I—specifically, the region consisting of six N-terminal amino acid residues (hereinafter referred to as "RI-1"), the region consisting of eight amino acid residues from domain I (hereinafter referred to as "RI-2"), and the region consisting of eight amino acid residues from domain I (hereinafter referred to as "RI-3")—with mouse sequences, the IL-36R message-specific blocking antibody of the present invention lost its binding activity to IL-36R. Therefore, it is important to presume these amino acid residues as antigenic determinants of the antibody, and by creating receptors that replace each of these residues with alanine, the antigenic determinants can be further identified. In Example 12, which evaluated these receptors, it was found that lysine (Lys or K) number 4 and isoleucine (Ile or I) number 28 were particularly important.

[0053] Since its discovery, IL-36R has been classified as part of the IL-1 family based on gene sequence homology. Because it possesses a TIR domain as an intracellular domain, it is believed that IL-1RAcP is essential for signal transduction, just like IL-1. Among the receptors belonging to the IL-1 family, the crystal structures of IL-1R1, IL-1R2, IL-18R, and IL-33R have been reported. Although the amino acid sequences of these receptors are not completely identical, they all possess three extracellular domains and have the same structure, thus forming a complex with ligands and IL-1RAcP (Non-Patent Document 10). Therefore, although the crystal structure of IL-36R has not yet been reported, it is believed that it has the same structural features as receptors belonging to the IL-1 receptor family. Homology modeling based on its crystal structure has been reported in several literatures, with the putative structure reported first (MAbs., (2017), 9[7]: 1143-54; J. Immunol., (2014), 193[2]: 921-30; J. Biol. Chem., (2016), 291

[32] : 16597-609; Sci. Rep., (2019), 9[1]: 9089).

[0054] Referring to these structures, it can be understood that the antigenic determinant sites of the IL-36R message-specific blocking antibody of the present invention, namely Lys (4th amino acid from number 1) and Ile (28th amino acid) of sequence number 11 (Pro001391), can block the messages of IL-36α and IL-36γ by binding the antibody to the region, even though they are far from the binding site of the IL-36 ligand. Although it is not clear why the message of IL-36β is not blocked, it is highly likely that the following reasons are true: the N-terminal region (region RI-1) which is highly likely to allow direct binding of the ligand, the circular domain region (region RI-2) starting with Ile (28th amino acid) located in a location structurally close to region RI-1, and the circular domain regions (regions RI-3) of numbers 81 to 88 interact with the antibody respectively, thereby generating message-specific blocking activity that blocks the messages caused by IL-36α and IL-36γ while not blocking the messages caused by IL-36β.

[0055] As explained above, the inventors have for the first time mastered the antigenic determinant of an IL-36R message-specific blocking antibody. By using the information of this antigenic determinant for screening, those skilled in the art can obtain antibodies against domain I of IL-36R, or antibodies against regions RI-1 to RI-3 and their surrounding regions, thereby efficiently obtaining the IL-36R message-specific blocking antibody of this invention without excessive trial and error. [IL-36R message-specific blocking antibody]

[0056] The IL-36R message-specific blocking antibody (appropriately referred to herein as "IL-36R message-specific blocking antibody of the present invention" or "antibody of the present invention") is an antibody that specifically binds to human IL-36R and, among the three human IL-36R agonist ligands IL-36α, β and γ, does not block the message of at least one ligand (preferably IL-36β), or the blocking of the message of that ligand is weaker than the blocking of the message of the other at least one ligand (preferably IL-36α and / or γ, more preferably both IL-36α and γ) (this property is appropriately referred to as "IL-36R message-specific blocking activity"). Here, the concept of relatively "weak" inhibition of the IL-36R signaling caused by the IL-36R agonist ligand (IL-36R agonist signaling) is explained by the larger value shown by the 50% inhibition concentration (IC50) of the IL-36R agonist signaling. Specifically, an antibody that inhibits the signaling caused by IL-36α and γ but not the signaling caused by IL-36β, or whose inhibition is weak, means that when the antibody is evaluated under the test conditions specified in Example 13 described later, the IC50 for the IL-36β signaling is greater than the IC50 for the IL-36α and γ signaling, preferably more than 10 times greater than the IC50 for the IL-36β signaling. More preferably, the IC50 for the IL-36β signaling caused by the antibody of the present invention is greater than 100 nM, and more preferably greater than 1 μM. On the other hand, the IC50 of the IL-36α and IL-36γ signals induced by the antibody of the present invention is expected to be less than 100 nM, then less than 10 nM, and then less than 1 nM.

[0057] Furthermore, the binding strength of the antibody to IL-36R in this invention is not particularly limited. The 50% effective concentration (EC50) of IL-36R measured in a Cell-based ELISA using IL-36R-expressing cells is generally preferably below 1×10⁻⁷ M or 1×10⁻⁸ M. Moreover, the "50% effective concentration" (EC50) of the antibody in this specification refers to the antibody concentration that indicates 50% of the maximum binding affinity of the antibody to the antigen. Specific measurement conditions for the EC50 of a Cell-based ELISA using IL-36R-expressing cells can be cited in the examples described below.

[0058] The antibody of the present invention does not have any particular limitation on its amino acid sequence, as long as it possesses the aforementioned IL-36R message-specific blocking activity. However, the antibody of the present invention preferably has specific amino acid sequences as each CDR sequence. Specifically, as described below. Furthermore, in this specification, the "identity" of the amino acid sequence refers to the proportion of identical amino acid residues, and the "similarity" refers to the proportion of identical or similar amino acid residues. The similarity and identity of the amino acid sequences can be determined, for example, by the BLAST method (the preset conditions of NCBI's PBLAST). Also, for example, when expressed as "more than 80% similarity", it is clear that it includes the case of "more than 80% identity".

[0059] Here, "similar amino acid residues" means amino acid residues having side chains that possess the same chemical properties (e.g., charge or hydrophobicity). Examples of similar amino acid residues include the following combinations.

[0060] (1) Amino acid residues with aliphatic side chains: glycine (Gly or G), alanine (Ala or A), valine (Val or V), leucine (Leu or L), and isoleucine (Ile or I) residues. (2) Amino acid residues with aliphatic hydroxyl side chains: serine (Ser or S) and threonine (Thr or T) residues. (3) Amino acid residues with acetylamine side chains: aspartic acid (Asn or N) and glutamine (Gln or Q) residues. (4) Amino acid residues with aromatic side chains: phenylalanine (Phe or F), tyrosine (Tyr or Y), and tryptophan (Trp or W) residues. (5) Amino acid residues with basic side chains: lysine (Lys or K), arginine (Arg or R), and histidine (His or H) residues. (6) Amino acid residues with acidic side chains: aspartic acid (Asp or D) and glutamic acid (Glu or E) residues. (7) Amino acid residues with sulfur-containing side chains: cysteine ​​(Cys or C) and methionine (Met or M) residues. Furthermore, the combination of (1) with methionine (Met or M) and the combination of (4) with histidine (His or H) residues are also considered as similar amino acid residues.

[0061] Furthermore, when a corresponding variant exists in the sequence of the reference germline, substitutions can be made without regard to similarity. The variant system of the reference germline includes substitution, deletion, or addition. Regarding substitution, this includes cases where amino acids that are presumed to affect the orientation of the side chain, such as proline (Pro or P) or glycine (Gly or G), can also be substituted.

[0062] One example of the antibody of the present invention may be an antibody comprising a heavy chain variable region having the following (1) to (3) CDR-H1 to H3 sequences.

[0063] (1) An amino acid sequence comprising (1a) sequence number 23 or 25 (XH001 or XH002) as a CDR-H1 sequence (preferably an amino acid sequence of sequence number 23 (XH001)), (1b) an amino acid sequence having at least 80% similarity (preferably identity) to the aforementioned amino acid sequence (1a), or (1c) an amino acid sequence in which one or two amino acid residues of the aforementioned amino acid sequence (1a) are substituted, deleted or added.

[0064] (2) includes (2a) an amino acid sequence selected from sequence numbers 27, 29, 31 and 213 (XH003~XH005 and XH007) as a CDR-H2 sequence (preferably the amino acid sequence of sequence number 27 (XH003)), (2b) an amino acid sequence having at least 88% similarity (preferably identity) to the amino acid sequence of (2a) above, or (2c) an amino acid sequence in which one or two amino acid residues of the amino acid sequence of (2a) above are substituted, deleted or added.

[0065] (3) includes (3a) an amino acid sequence selected from sequence numbers 33, 215 and 217 (XH006, XH008 and XH009) as a CDR-H3 sequence (preferably the amino acid sequence of sequence number 33), (3b) an amino acid sequence having at least 83% similarity (preferably identity) to the amino acid sequence of (3a) above, or (3c) an amino acid sequence in which one or two amino acid residues of the amino acid sequence of (3a) above are substituted, deleted or added.

[0066] One example of the antibody of the present invention may be an antibody comprising a light chain variable region having the following (1) to (3) CDR-L1 to L3 sequences.

[0067] (1) An amino acid sequence comprising (1a) any one of sequence numbers 35, 37 and 219 (XL001, XL002 and XL006) as a CDR-L1 sequence (preferably the amino acid sequence of sequence number 35 (XL001)), (1b) an amino acid sequence having at least 84% similarity (preferably identity) to the amino acid sequence of (1a) above, or (1c) an amino acid sequence in which one or two amino acid residues of the amino acid sequence of (1a) above are substituted, deleted or added.

[0068] (2) includes (2a) an amino acid sequence selected from sequence numbers 39, 41 and 221 (XL003, XL004 and XL007) as a CDR-L2 sequence (preferably the amino acid sequence of sequence number 39 (XL004)), (2b) an amino acid sequence having at least 85% similarity (preferably identity) to the aforementioned amino acid sequence (2a), or (2c) an amino acid sequence in which one or two amino acid residues of the aforementioned amino acid sequence (2a) are substituted, deleted or added.

[0069] (3) includes (3a) an amino acid sequence selected from sequence numbers 43, 223 and 225 (XL005, XL008 and XL009) as a CDR-L3 sequence (preferably the amino acid sequence of sequence number 43), (3b) an amino acid sequence having at least 88% similarity (preferably identity) to the amino acid sequence of (3a) above, or (3c) an amino acid sequence in which one or two amino acid residues of the amino acid sequence of (3a) above are substituted, deleted or added.

[0070] One form of the antibody of the present invention can be an antibody obtained by combining a heavy chain variable region having any of the aforementioned CDR-H1~H3 sequences and a light chain variable region having any of the aforementioned CDR-L1~L3 sequences.

[0071] One example of the antibody of the present invention may be an antibody having any of the following (a) to (c) as the heavy chain variable region sequence.

[0072] (a) An amino acid sequence selected from any one of the following: sequence number 45 (Pro000713v), sequence number 47 (Pro000722v), sequence number 49 (Pro001558v), sequence number 51 (Pro001562v), sequence number 53 (Pro001554v), sequence number 55 (Pro001566v), sequence number 57 (Pro001570v), sequence number 59 (Pro001574v), sequence number 177 (Pro002817v), sequence number 181 (Pro002818v), and sequence number 185 (Pro002819v) (preferably the amino acid sequence of sequence number 45 (Pro000713v)).

[0073] (b) An amino acid sequence having at least 80% (preferably 85%, 90%, 95%, 96%, 97%, 98%, or 99%) similarity (preferably identity) to the amino acid sequence of (a) above.

[0074] (c) An amino acid sequence in which 1 to 12 (preferably 1 to 11, more preferably 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2 or 1) amino acid residues in the amino acid sequence of (a) are substituted, deleted or added.

[0075] One example of the antibody of the present invention may be an antibody having any of the following (a) to (c) as the light chain variable region sequence.

[0076] (a) An amino acid sequence selected from any one of the following: sequence number 61 (Pro000714v), sequence number 63 (Pro001332v), sequence number 65 (Pro001333v), sequence number 67 (Pro001334v), sequence number 179 (Pro002821v), sequence number 183 (Pro002823v), and sequence number 187 (Pro002822v) (preferably the amino acid sequence of sequence number 61 (Pro000714v)).

[0077] (b) An amino acid sequence having at least 80% (preferably 85%, 90%, 95%, 96%, 97%, 98%, or 99%) similarity (preferably identity) to the amino acid sequence of (a) above.

[0078] (c) An amino acid sequence in which 1 to 10 (preferably 1 to 9, more preferably 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2 or 1) amino acid residues in the amino acid sequence of (a) are substituted, deleted or added.

[0079] One form of the antibody of the present invention can be an antibody obtained by combining any of the aforementioned heavy chain variable regions and any of the aforementioned light chain variable regions respectively.

[0080] Furthermore, methods for identifying the sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, or CDR-L3 in antibodies can be exemplified by, for example, the Kabat method (NIH Publication, (1991), No. 91-3242) or the Chothia method (J. Mol. Biol., (1997), 273[4]: 927-48). These methods are common knowledge to those skilled in the art, and their outlines can be found, for example, on the homepage of Dr. Andrew CR Martin's Group website (http: / / www.bioinf.org.uk / abs / ). Furthermore, the CDR in this invention is described as the CDR of Kabat.

[0081] The frame sequences of the heavy chain variable region and light chain variable region of the immunoglobulin of the antibody of the present invention are preferably frame sequences of various types of vertebrate immunoglobulins. More preferably, they are frame sequences of various types of human or non-human animal immunoglobulins, including mice or rats.

[0082] By appropriately combining the amino acid sequences of the CDRs and / or variable regions of the heavy and light chains of human or non-human animal antibodies (including mice or rats) with the amino acid sequences of the framework regions and / or constant regions of the heavy and light chains, those skilled in the art can design the IL-36R signaling-specific blocking antibody of the present invention. In particular, by using the amino acid sequences of the framework regions and / or constant regions of the heavy and light chains of human antibodies, a humanized IL-36R signaling-specific blocking antibody can be formed. The amino acid sequences of the framework regions and / or constant regions of the heavy and light chains of the humanized antibody can be selected, for example, from various types or variants of human IgG, IgA, IgM, IgE, IgD.

[0083] Furthermore, for reference, the amino acid sequence of the heavy chain constant region of rat antibody example 117 (Experimental Example 1, C_00028) is shown in sequence number 159, and the base sequence of the gene encoding it is shown in sequence number 160. Also, the heavy chain constant region of a general human IgG1 antibody is shown in sequence number 161, and the base sequence of the gene encoding it is shown in sequence number 162. Furthermore, the heavy chain constant region of example B3 of the humanized antibody described in the prior art literature, and example 117hG1Dk of a rat / human chimeric antibody in which the constant region of the antibody of Experimental Example 1 is replaced with a human-type antibody, is shown in sequence number 163, and the base sequence of the gene encoding it is shown in sequence number 164. Furthermore, the heavy chain constant region of the humanized antibody example APE6060 described in previous technical documents, and the heavy chain constant region of the rat / human chimeric antibody example 117hG4k (which replaces the constant region of the antibody in Example 1 with a human type), are shown in sequence number 165, and the base sequence of the gene encoding it is shown in sequence number 166. Also, the amino acid sequence of the light chain constant region of the rat antibody example 117 is shown in sequence number 167, and the base sequence of the gene encoding it is shown in sequence number 168. Furthermore, the amino acid sequences of the human light chain constant region of chimeric antibodies 117hG1Dk and 117hG4k, the humanized antibody example B3, and APE6060 are shown in sequence number 169, and the base sequence of the gene encoding it is shown in sequence number 170.

[0084] The antibody of the present invention is preferably of the IgG type or a variant thereof, more preferably of the human IgG type or a variant thereof, the human IgG4 subtype or a variant thereof, or the human IgG1 subtype or a variant thereof. In one example, the stabilized IgG4 constant region, according to the Kabat system, contains proline at position 241 of the hinge region. This position, according to EU numbering (Proc. Natl. Acad. Sci. USA, (1969), 63[1]: 78-85) and relevant immunological protein sequences (Washington DC United States Department of Health and Human Services, 2001 and NIH Publication, (1991), No. 91-3242), corresponds to position 228 of the hinge region. In human IgG4, this residue is generally serine, and stabilization can be induced by replacing serine with proline. In one example, the N297A variant was introduced into a constant region of IgG1 to suppress binding to the Fc receptor and / or fixation of complement as much as possible. [Competitive binding]

[0085] One aspect of the present invention includes antibodies that competitively bind to the IL-36R signal-specific blocking antibody of the present invention. Such competitively binding antibodies are also included within the scope of the present invention. In the present invention, "competitive binding" means the phenomenon where, when multiple monoclonal antibodies coexist with an antigen, the binding of one antibody to the antigen is blocked by the binding of another antibody to the antigen. Generally, this can be determined by adding a certain amount (concentration) of monoclonal antibody while varying the amount (concentration) of other monoclonal antibodies, and measuring the amount (concentration) of added monoclonal antibodies that reduces the binding of a certain amount of the former monoclonal antibody to the antigen. The degree of blocking can be expressed as IC50 or Ki values. Monoclonal antibodies that competitively bind to the IL-36R signal-specific blocking antibody of the present invention refer to antibodies whose IC50 is typically below 1000 nM, particularly below 100 nM, and further below 10 nM, when using 10 nM of the IL-36R signal-specific blocking antibody of the present invention to detect antigen-antibody binding. When performing competitive binding assays, the antibodies used can be labeled with enzymes, fluorescent substances, luminescent substances, radioactive isotopes, etc., and detected using assay methods suitable for the physical and / or chemical properties of the labeled substance. Alternatively, biosensors such as surface plasma resonance (SPR) or bio-layer interferometry (BLI) can be used. [Manufacturing method of IL-36R signal-specific blocking antibody]

[0086] The antibody of the present invention can be obtained using techniques known to those skilled in the art. The antibody of the present invention is a multi-strain antibody or a monoclonal antibody (Nature, (1983), 305(5934): 537-40). For example, a multi-strain antibody can be obtained by an IL-36R protein having an amino acid sequence represented by sequence number 11 (Pro001391), or an IL-36R partial peptide having a portion thereof (e.g., a peptide comprising domain I formed by amino acid residues 1 to 100, or comprising a RI-1 region formed by 6 residues on the N-terminal side of the aforementioned domain I, a RI-2 region formed by 8 residues of amino acids 25 to 32, and / or a region formed by residues 81 to 88 of domain I). An antigen can be a peptide containing the amino acid sequence of the RI-3 region (8 residues of the amino acid of the IL-36R protein) or an antigenic polynucleotide encoding a portion of the IL-36R protein (Sequence No. 13: Q9HB29|ILRL2_HUMAN), or administered intramuscularly or subcutaneously to mammals to sensitize them by antigen expression, and then recovered from the animal's serum. Furthermore, when using peptides as antigens, antigens in the form of proteins or polyisocyanates bound to BSA or KLH can be used.

[0087] The monoclonal antibody of this invention can be obtained by administering IL-36R nucleotides with the amino acid sequence represented by sequence number 11 (Pro001391) to a mammal, causing antigen production in the animal, and then taking immune cells from the animal sensitized with the produced antigen, fusing them with myeloma cells, etc., and selecting the resulting fusion tumors, which are then recovered from their cultures. Such methods of obtaining monoclonal antibodies (Nature, (1992), 356

[6365] : 152-4; Patent Document 4) or cell fusion methods (Nature, (1975), 256

[5517] : 495-7) have been reported and generalized, and the efficiency of obtaining them can be further improved by administering immune-activating substances (Cancer Gene Ther., (2007), 14

[11] : 904-17). Monoclonal antibodies obtained thereby may include, but are not limited to, heavy chains having a heavy chain variable sequence (heavy chain variable region) represented by sequence number 45 (Pro000713v) and light chains having a light chain variable sequence (light chain variable region) represented by sequence number 61 (Pro000714v).

[0088] The obtained monoclonal antibody can also be used to obtain a nucleic acid molecule having a gene sequence encoding the amino acid sequence of the heavy chain and light chain proteins constituting the antibody. One example of a method for obtaining such a gene sequence is not limited, but the method described in the following examples can be cited. The monoclonal antibody obtained thereby is not limited, but examples include antibodies composed of a heavy chain having the amino acid sequence number 77 and a light chain having the amino acid sequence number 79 (C_00028; CL_00071).

[0089] Antibodies can also be genetically engineered using nucleic acid molecules encoding amino acids of proteins that constitute such antibodies, such as heavy and light chains. As genetic information for the antibody, changes are made to the H and L chains, or sequence information such as variable regions or CDRs, to enhance the antibody's binding or specificity. Alternatively, antibodies from animals such as mice can be modified into human-type antibodies to create antibodies with structures suitable for therapeutic use. This is a technique well-known to those skilled in the art. Furthermore, human-type monoclonal antibodies can be obtained by using non-human genetically modified animals with introduced human antibody genes as antigen-sensitizing animals. In addition, the use of a phage gene library that expresses a variable region or a portion thereof representing a human antibody (human antibody phage display) as a method that does not require sensitization of animals to obtain phage strains composed of antibodies that specifically bind to the corresponding antigen or specific amino acid sequences, and the production of human antibodies from this information, is also a technique that can be appropriately performed by those with ordinary knowledge in the relevant technical field (e.g., see the review of Keio J. Med., (2011), 60:37-46).

[0090] Furthermore, as a method for manufacturing the aforementioned monoclonal antibody, fusion tumors that produce the desired antibody can be cultured separately, and the antibody can be purified from the obtained culture supernatant using conventional methods. Alternatively, as another manufacturing method, the gene encoding the antibody, more specifically the gene encoding the heavy chain and / or light chain of an immunoglobulin, can be obtained from the fusion tumor that produces the desired antibody or from a phage strain obtained by displaying a human antibody phage. A vector for expressing this gene can be prepared and introduced into a host cell (mammalian cell, insect cell, microorganism, etc.) to produce the antibody. In this case, the gene modification to introduce the desired properties into the gene encoding the heavy chain and / or light chain of the immunoglobulin, and the use of structural information of the variable region or CDR region of the heavy chain and / or light chain of the immunoglobulin to produce human-typed antibodies, antibody chimeric proteins, low-molecular-weight antibodies, or scaffold antibodies, can be implemented by those skilled in the art using known techniques. Furthermore, to improve antibody performance or avoid side effects, alterations can be made to the structure of constant regions of the antibody or to modify the glycan portion, which can be done appropriately using techniques known to those with ordinary knowledge in the relevant technical field.

[0091] The IL-36R message-specific blocking antibody of the present invention can be obtained using techniques known to those skilled in the art. Specifically, the IL-36R message-specific blocking antibody of the present invention is usually a monoclonal antibody (Nature, (1975), 256

[5517] : 495-7), which can be prepared, for example, by the following methods.

[0092] For example, a nucleic acid molecule is prepared containing the amino acid sequence of the heavy and / or light chains of an immunoglobulin encoding the IL-36R signal-specific blocking antibody of the present invention. Here, a carrier or plasmid containing the nucleic acid molecule can also be prepared by introducing the nucleic acid molecule into various carriers or plasmids. Next, the host cell is transformed using the aforementioned nucleic acid molecule, carrier, or plasmid. Examples of host cells include eukaryotic cells such as mammalian cells, insect cells, yeast cells, or plant cells, or bacterial cells. Next, the transformed host cell is cultured under appropriate conditions for producing the IL-36R signal-specific blocking antibody of the present invention. Here, the obtained IL-36R signal-specific blocking antibody of the present invention can also be isolated from the host cell as needed. Various methods used in these sequences are well known to those skilled in the art.

[0093] Furthermore, as a method for sensitizing animals, a non-human genetically modified animal with an introduced human antibody gene can be used as the animal to sensitize the antigen. IL-36R and / or some of its peptides are sensitized, immune cells are extracted and fused with myeloma cells, etc., and the resulting fusion tumor is selected and colonized. The resulting culture supernatant is then purified and recovered using conventional methods to obtain the antibody. Such a method for obtaining a monoclonal antibody is described, for example, in Patent Document 4.

[0094] Alternatively, a phage gene library (human antibody phage display) that utilizes the variable region or a portion thereof that expresses the desired humanized antibody can be used to obtain a phage strain composed of an antibody that specifically binds to the corresponding antigen or a specific amino acid sequence, and a technique for producing humanized antibodies from that information can be used (e.g., see the review of Keio J. Med., (2011), 60:37-46).

[0095] Here, gene alterations are made to the genes encoding the heavy and / or light chains of immunoglobulins to introduce desired properties, or structural information on variable regions or CDR regions of the heavy and / or light chains of immunoglobulins is used to produce antibody chimeric proteins, low-molecular-weight antibodies, scaffold antibodies, etc., which can be implemented using known techniques by those skilled in the art. Furthermore, modifications are made to the structure of constant regions of antibodies, or to portions of the glycans, for the purpose of improving antibody performance or avoiding side effects; these can also be appropriately performed by those skilled in the art using well-known techniques. [Drugs containing IL-36R signal-specific inhibitory antibodies]

[0096] The IL-36R signaling-specific blocking antibody of the present invention can be used as an active ingredient in medicines (pharmaceutical compositions) for treating and / or preventing IL-36-related states or diseases caused by the action of IL-36R (IL-36 receptor). Furthermore, in this specification, "pharmaceutical" and "pharmaceutical composition" are used synonymously. Specifically, conditions associated with IL-36 or IL-36R, or diseases that specifically inhibit antibody treatment or prevention using IL-36R signals, include autoimmune diseases such as psoriasis vulgaris, pustular psoriasis (generalized), psoriatic erythroderma, psoriatic arthritis, palmoplantar pustulosis, ulcerative colitis, Crohn's disease, systemic lupus erythematosus, lupus nephritis, asthma, atopic dermatitis, transplant rejection, generalized rigidity, type 1 diabetes mellitus, optic neuritis, uveitis, stroke, schizophrenia, amyotrophic lateral sclerosis, dermatomyositis, ankylosing spondylitis, polymyositis, pulmonary fibrosis, Hashimoto's thyroiditis, myasthenia gravis, autoimmune thyroiditis, Behcet's disease, and Basedow's disease. Diseases that could be considered as cancer treatments include neuroblastoma, rheumatoid arthritis, multiple sclerosis, arthritis, Sjögren's syndrome, and cytokine-releasing syndrome. Diseases for which this drug could be considered as a treatment include neuroblastoma, rhabdomyosarcoma, osteosarcoma, pediatric cancer, ovarian cancer, pancreatic cancer, breast cancer, prostate cancer, bone cancer, lung cancer, colorectal cancer, neck cancer, synovial sarcoma, bladder cancer, gastric cancer, Wilms' tumor, metastatic carcinoid tumors, and diarrhea associated with vasoactive intestinal peptide-secreting tumors, VIP tumor, Verner-Morrison syndrome, Beckwith-Wiedemann syndrome, kidney cancer, renal cell carcinoma, transitional cell carcinoma, and Ewing's sarcoma. Sarcoma), leukemia, acute lymphoblastic leukemia, brain tumors, glioblastoma, non-glioblastoma brain tumors, spinal cord meningioma, pituitary adenoma, vestibular schwannoma, undifferentiated neuroectodermal tumors, medulloblastoma, astrocytoma, oligodendroglioma, ependymoma, choroid plexus papilloma.

[0097] The IL-36R neutralizing antibody (IL-36R signaling-specific blocking antibody) of the present invention is preferably used as a therapeutic or preventative agent for diseases classified as psoriasis and a phenotype of psoriasis. Furthermore, the IL-36R signaling-specific blocking antibody of the present invention, by inhibiting inflammation associated with the rise of IL-36α or IL-36γ and without inhibiting IL-36β, is expected to have excellent effects on diseases where resistance to bacteria promotes therapeutic efficacy, such as Crohn's disease or atopic dermatitis. Moreover, compared to PAN antibodies, this antibody is less likely to cause side effects due to viral infections such as herpes zoster, and is less likely to produce intradermal growths or malignant growths.

[0098] The medicines that regulate or modify immune responses in this invention refer to immunomodulatory drugs, etc. Immunomodulatory drugs improve symptoms by regulating abnormal immune responses and inhibiting the production of substances in the body that are factors causing inflammation. These medicines have the effect of inhibiting the production of substances such as immunoglobulins or cytokines, suppressing the progression of the disease, or alleviating its symptoms. Specifically, these medicines regulate the amplitude and duration of immune and inflammatory responses at sites of inflammation or allergic immune responses caused by cancer, autoimmune diseases, infections, microbial toxins, inflammatory agents, and allergic reactions. Abnormal regulation of excessive IL-36 production or expression can lead to disease states. Therefore, IL-36 is associated with various inflammatory and immunomodulatory diseases and symptoms, and systemic or local excess of IL-36 is a cause of abnormal immune responses. Medicines that block IL-36-induced information are expected to act as substances that regulate immune responses.

[0099] The drug containing the IL-36R signaling-specific blocking antibody of the present invention can also be formulated as a pharmaceutical composition containing, in addition to the IL-36R signaling-specific blocking antibody of the present invention, a pharmaceutically permissible drug carrier and / or other additives. Formulations using pharmaceutically permissible drug carriers and / or other additives can be carried out, for example, by the method described in University of the Sciences in Philadelphia, “Remington: The Science and Practice of Pharmacy, 20th EDITION”, Lippincott Williams & Wilkins, (2000). One form of such a therapeutic or preventative agent is supplied as a liquid or lyophilized agent prepared by dissolving, suspending, or emulsifying in a sterile aqueous or oily liquid. Such solvents or solutions, in the case of aqueous solutions, include distilled water for injection and physiological saline. When osmotic pressure regulators (such as D-glucose, D-sorbitol, D-mannitol, sodium chloride, etc.) are further added, appropriate dissolving aids such as alcohols (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., polysorbate 80, polyoxyethylene cured castor oil 50) are also used. Furthermore, oily solutions are also used as solvents or solutions, such as sesame oil and soybean oil, and sometimes benzoate, benzyl alcohol, etc., are used as dissolving aids. Such formulations may contain additives such as buffers (e.g., phosphate buffers, acetate buffers), analgesics (e.g., benzalkonium chloride, procaine hydrochloride), stabilizers (e.g., human serum albumin, polyethylene glycol), preservatives (e.g., ascorbic acid, isoascorbic acid and their salts), colorants (e.g., copper chlorophyll, β-carotene, Red No. 2, Blue No. 1), preservatives (e.g., parabens, phenols, benzethonium chloride, benzalkonium chloride), thickeners (e.g., hydroxypropyl cellulose, carboxymethyl cellulose and their salts), stabilizers (e.g., human serum albumin, mannitol, sorbitol), and odorants (e.g., menthol, citrus flavorings).

[0100] Furthermore, as other forms of therapeutic or preventative agents, examples include powders, lozenges, granules, capsules, pills, suppositories, tablets, and other solid dosage forms. When the solid dosage form is administered orally, excipients (e.g., crystalline cellulose, lactose, starch), lubricants (e.g., magnesium stearate, talc), binders (e.g., hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyethylene glycol), and disintegrants (e.g., starch, calcium carboxymethyl cellulose), etc., may be used as additives. Additionally, preservatives (e.g., benzyl alcohol, chlorobutanol, methylparaben, propylparaben), antioxidants, colorants, sweeteners, etc., may be used as needed. Furthermore, other forms of therapeutic or preventative agents applicable to mucous membranes can also be listed. These formulations may contain adhesives, thickeners, and viscous agents (such as mucin, agar, gelatin, pectin, carrageenan, sodium alginate, locust bean gum, senna gum, tragacanth gum, gum arabic, chitosan, pullulan, glutinous starch, sucralfate, cellulose, and their derivatives) as additives, primarily intended to impart adsorption and retention properties to mucous membranes. However, the form, solvent, or additives of therapeutic or preventative agents supplied to living organisms are not limited to these; those skilled in the art can make appropriate selections.

[0101] A drug containing the IL-36R neutralizing antibody of the present invention may also contain other existing drugs (active ingredients) besides the IL-36R neutralizing antibody of the present invention. Furthermore, a drug containing the IL-36R signal-specific blocking antibody of the present invention may also be combined with other existing drugs as a package. Examples of active ingredients that can be combined with the IL-36R signal-specific blocking antibody include IL-36Ra, IL-38, immunomodulatory drugs or analogues, immunosuppressants or analogues, antibacterial drugs or analogues, anticancer agents or analogues, IL-36R signal-specific blocking antibodies or analogues, azelastine, oxatomide, mequitazine, fexofenadine, epinastine, ebastine, and cetirizine. Levocetirizine, bepotastine, emedastine, olopatadine, loratadine, levocabastine, ozagrel, seratrodast, ramatroban, pranlukast, montelukast, zafirlukast, subplatast, diphenhydramine, dimenhydrinate, diphenylpyraline, clemastine, chlorpheniramine, triprolidine, promethazine, alimemazine, hydroxyzine, homochlorcy clizine), cyproheptadine, mesalazine, interferon beta-1b, interferon beta-1a, fingolimod hydrochloride, natalizumab, glatiramer acetate, dimethyl fumarate, tacrolimus, mercaptopurine, azathioprine, apremilast, corticosteroids, antiemetics, ondansetron hydrochloride.Granisetron hydrochloride, Metroclopramide, Domperidone, Haloperidol, Cycizine, Lorazepam, Prochlorperazine, Dexamethasone, Levomepromazine, Tropisetron, Cancer vaccines, GM-CSF inhibitors, GM-CSF, DNA vaccines, Cell-based vaccines, Dendritic cell vaccines, Recombinant virus vaccines, Heat shock protein (HSP) vaccines, Homologous tumor vaccines, Autologous tumor vaccines, Analgesics, Ibuprofen, Naproxen, Magnesium trisalicylate choline, Oxycodone hydrochloride, Antiserum Anti-inflammatory drugs, antithrombotic drugs, anti-PD-1 antibodies, nivolumab, pembrolizumab, tislelizumab, anti-PD-L1 antibodies, atezolizumab, avelumab, durvalumab, anti-CTLA4 antibodies, ipilimumab, anti-CD20 antibodies, rituximab, anti-HER2 antibodies, trastuzumab, anti-CCR4 antibodies, mogamulizumab, anti-VEGF antibodies, bevacizumab, anti-VEGF receptor antibodies, soluble VEGF receptor fragments, anti-TWEAK antibodies, anti-TWEAK receptor antibodies, soluble TWEAK receptor fragments, AMG 706, AMG 386, anti-α4β7 antibody, bevacizumab, etrolizumab, anti-SIRPα antibody, anti-CD40 antibody, anti-CD40L antibody, anti-proliferative drugs, farnesyl protein transferase inhibitors, αvβ3 inhibitors, αvβ5 inhibitors, p53 inhibitors, Kit receptor inhibitors, ret receptor inhibitors, PDGFR inhibitors, growth hormone secretion inhibitors, angiopoietin inhibitors, tumor-infiltrating macrophage inhibitors, c-fms inhibitors, anti-c-fms antibodies, CSF-1 inhibitors, anti-CSF-1 antibodies, soluble c-fms fragments, pegvisomant, gemcitabine, panitumumab, irinotecan.Etretinate, phosphodiesterase 4 (PDE4) inhibitors, TNFα inhibitors, IL-17A inhibitors, IL-17F inhibitors, IL-17RA inhibitors, IL-23p19 inhibitors, IL-23p40 inhibitors, and IL-1RAcP inhibitors, SN-38. The dosage of drugs other than the IL-36R signaling-specific inhibitory antibody can be the usual therapeutic dosage, but may be increased or decreased depending on the situation.

[0102] The therapeutic or preventative agent of the present invention can be administered non-orally for the purpose of symptom improvement. In cases of non-oral administration, it may be a nasal preparation, such as a liquid, suspension, or solid formulation. Other non-oral forms may include injections, such as subcutaneous, intravenous, drip, intramuscular, intraventricular, or intraperitoneal injections. Other non-oral preparations may include suppositories, sublingual, transdermal, and mucosal administration methods other than nasal administration. Furthermore, it may be administered locally within blood vessels in the form of a stent or endovascular embolization agent. It may also be administered, depending on the circumstances, in the form of DNA or RNA encoding the antibody, or in the form of cells or indigenous bacteria producing the antibody.

[0103] The dosage of the therapeutic or preventive agent in this invention varies depending on the patient's age, sex, weight, symptoms, treatment effect, administration method, treatment time, or the type of active ingredient contained in the pharmaceutical composition. Generally, for adults, the main agent can be administered at a dose ranging from 0.1 mg to 1 g, preferably from 0.5 mg to 300 mg, once every 1 to 4 weeks, or once every 1 to 6 months. However, the dosage and frequency of administration may vary depending on various conditions. Therefore, there are cases where a dosage and frequency lower than those mentioned above are sufficient, and there are also cases where a dosage and frequency exceeding those ranges are necessary. Furthermore, the therapeutic or preventive agent in this invention, by reducing side effects, can achieve effects with a short administration period or be administered long-term. [Example]

[0104] The present invention will be further described in detail below with reference to embodiments, but these embodiments are merely examples shown for the convenience of illustration, and the present invention is not limited to these embodiments in any sense.

[0105] [Example 1] Formulation of hIL-36R gene, etc. The gene encoding human IL-36R (hIL-36R) (Sequence No. 11: Pro001391) was designed based on the reference sequence NM_003854.4 of the mRNA of IL1RL2 interleukin 1 receptor like 2 [Homo sapiens (human)] registered in NCBI. Specifically, the gene contains the amino acid sequence encoding hIL-36R, the EcoRI and KOZAK sequences configured at its 5' end, and the NotI recognition sequence configured at its 3' end, and was formulated artificially (Sequence No. 171: DNA-000224S). The synthesized gene was selected and colonized into pIRESpuro3 (Chrontech Cat. No.). 631619, sequence number 172: vDNA-000011) and pCAG-Neo (Wako Pure Chemicals Cat. No. 163-25601, sequence number 173: vDNA-000005). These are respectively referred to as hIL-36R expressor 1 (selected from pIRESpuro3) and hIL-36R expressor 2 (selected from pCAG-Neo). Furthermore, the gene encoding human IL-1RAcP (sequence number 174: NM_002182) and the gene encoding mouse IL-1RAcP (sequence number 175: NM_008364) are also endowed with 5' end... EcoRI and KOZAK sequences, along with a NotI sequence attached to the 3' end, were artificially synthesized and subcloned into pIRESpuro3. The plastid containing the NF-kB reporter gene of the firefly luciferase gene used in the reporter gene analysis was pGL4.32 (Promega, E8491) (hereinafter referred to as the "reporter plastid"). The plastid used as the control group reporter plastid for the Renilla luciferase gene correction was a gene encoding the promoter (sequence number 176: vDNA-000028) introduced into the commercially available plastid pGL4.70 (Promega, E6881) using a restriction enzyme reaction (hereinafter referred to as the "correction plastid").

[0106] [Example 2] Preparation of CHO cells stably expressing hIL-36R Cells stably expressing human IL-36R were prepared by transfecting CHO-K1 cells (JCRB cell bank, JCRB9018) with hIL-36R expression plastids 1 using lipid transfection. A stable cell line was then prepared using Ham's F12 medium supplemented with appropriate concentrations of puromycin and serum. The stable cell lines prepared here were used in Cell ELISA in Examples 6, 9, and 15, and in flow cytometry in Example 4, as described later.

[0107] [Example 3] Sufficient 293A cells (R70507, Thermofisher Scientific) were prepared for the functional evaluation of hIL-36R using electroporation. Cells were cultured in 245mm × 245mm square cell culture dishes using a confluent method during electroporation. Cells used for electroporation were removed from the culture dishes with trypsin-EDTA solution, and recovered after adding DMEM medium supplemented with 10% FBS. An appropriate amount of cell suspension was transferred to a 50mL tube and centrifuged at 300g × 5 minutes, 4°C to prepare cell clumps. After removing the culture medium, 10mL of electroporation buffer was added to each tube to resuspend the cells, and the number of viable cells was counted. The cell suspensions suspended in electroporation buffer were combined and further centrifuged at 300g × 5 minutes, 4°C, and then resuspended in electroporation buffer to achieve a viable cell count of 1 × 10⁸ cells / mL. For each electroporation cycle, 400 μL of cell suspension was used. 25 μg of hIL-36R elicitor plasmid 1, 25 μg of reporter plasmid, and 10–25 μg of corrective plasmid were added at a concentration of 3 mg / mL or higher. The mixture was carefully mixed using a pipette. The cell suspension was then transferred to a 400 μL cartridge, and electroporation was performed using Maxcyte STX (registered trademark) according to the 293 Cell Laboratory Protocol. After each electroporation cycle, the cells were carefully transferred to one well of a 24-well tray using a pipette and incubated at 37°C and 5% CO2 for 30 minutes.

[0108] After standing, the cells were recovered with culture medium and cultured in square cell culture dishes for 1 day. The recovered cells were recovered again with trypsin-EDTA solution and culture medium, and then cryopreserved at an appropriate concentration using cell preservation solution. Cell preparation was confirmed using the Promega Dual-Glo (registered trademark) Luciferase Assay System (E2920 or E2940, Promega). The cryopreserved cells were thawed, resuspended in 10 mL of culture medium, centrifuged at 300 g × 5 min at 4 °C to break up cell clumps, the preservation agent was removed, and the cells were resuspended in 10 mL of culture medium. The number of viable cells was counted. A cell suspension of 2 × 10⁵ cells / mL was prepared and seeded in 96-well Optical Corning (registered trademark) BioCoat (registered trademark) poly-D-lysine 96-well white flat bottom microplates, making a volume of 100 μL / well, and cultured until the next day. After removing the culture medium, 30 μL of the culture medium used for culturing fusion tumors was added, and the mixture was incubated at 37°C and 5% CO2 for 30 minutes. Then, a series of dilutions of each ligand of active human IL-36α (6995-IL / CF, manufactured by R&D Company), human IL-36β (6834-ILB / CF, manufactured by R&D Company), and human IL-36γ (6835-IL / CF, manufactured by R&D Company) at an appropriate concentration of 200 ng / μL were added to each well, making 30 μL / well, and incubated at 37°C and 5% CO2 for 5 hours. Furthermore, wells in the aforementioned dish were further set up as a negative control group (containing only culture medium and no ligands) and as a positive control group (consisting of a combination of culture medium and ligand solutions).

[0109] After 5 hours, remove the culture medium and add 100 μL / well of Dual-Glo (registered trademark) fluorescent enzyme assay reagent diluted 2-fold with PBS. Shake gently for 10 minutes in the dark, and measure using a Tristar LB941 (Berthold Technologies) with "Counting Time" set to "0.1 sec" and "Emission Filter" set to "No filter slot-A8". Record the measured value as the F-value. Label the F-value of any well as Fx, the average F-value at the highest ligand concentration as Fposi, and the average F-value of the negative control group as Fnega.

[0110] Next, add diluted Dual-Glo (registered trademark) Stop & Glo (registered trademark) reagent to a concentration of 50 μL / well, and gently shake for 10 minutes while shielded from light. The measurement is then performed in the same manner. This measured value is the R value. The R value of any well is labeled Rx, the average R value of the highest ligand concentration is labeled Rposi, and the average R value of the negative control group is labeled Rnega.

[0111] The message of NFkB is defined as follows.

[0112] The antibodies were evaluated using cells that had an F value of 1000 or higher and an R value of 100 or higher after the above experiment was performed using human IL-36α ligand at a final concentration of 100 ng / mL.

[0113] [Example 4] Confirmation of Human IL-36R Expression and Functional Evaluation of CHO Cells Stably Expressing Human IL-36R The expression of human IL-36R and human IL-1RAcP in CHO cells stably expressing human IL-36R and cells used for human IL-36R functional evaluation were recovered from culture dishes and reacted with diluted anti-hIL-36R antibody (R&D, AF872) at room temperature for 20 minutes. After washing once with Cell Staining Buffer (BioLegend, 420201), the cells were reacted with a 1000-fold diluted anti-goat IgG-Alexa Fluor (registered trademark) 488 labeling antibody (secondary antibody) (Thermo Fisher Scientific, A-11078) for 20 minutes to detect anti-human IL-36R antibody. After washing once, the cells were resuspended to an appropriate concentration and measured by flow cytometry using a BD Accuri C6 (Becton Dickinson) instrument. The detected cells were gated using both forward scatter (FSC) and side scatter (SSC) to confirm the expression of human IL-36R (CHO cells stably expressing human IL-36R: Figure 5A; cells for functional evaluation: Figure 5C). Furthermore, the negative control group consisted of cells that did not elicit a single antibody response to cells expressing human IL-36R, but only a two-stage antibody response. Additionally, the expression of human IL-1RAcP in 293A cells and cells for functional evaluation was similarly detected by flow cytometry using an anti-IL-1RAcP antibody (R&D, AF676) (293A cells: Figure 5B; cells for functional evaluation: Figure 5D).

[0114] [Example 5] Preparation of fusion tumors producing rat anti-human IL-36R monoclonal antibodies: Immunotherapy of rats with human IL-36R was performed using DNA immunization with a gene gun. Flow cytometry (FCM) was used to measure antibody titers in the blood after collection. For all individuals whose antibody titers increased from the start of DNA immunization to approximately 6 weeks later, a boost was performed. Lymphoid cells were recovered from the animals and cryopreserved using cell freezing solution.

[0115] Rat anti-human IL-36R monoclonal antibody was obtained by electrofusion to create fusion tumors. Specifically, 1.8 × 10⁷ P3U1 cells and rat lymphocytes were suspended in 700 μL of ECF buffer (0.3 M mannitol, 10 mM CaCl₂, 10 mM MgCl₂, 1 mg / mL BSA). Using a cell fusion device (ECFG21, Nepa Gene) and a chamber electrode (CUY497P2), the cells were fused by applying an AC voltage of 30 V for 10 seconds and a DC voltage of 350 V for 30 μ seconds × 3 times. The fused cells were then suspended in a selective medium containing hypoxanthine, aminopterin, and thymidine, seeded in 96-well plates containing more than 200 μL of culture medium, and cultured.

[0116] [Example 6] Screening for Fusculomas Produced by Anti-human IL-36R Antibodies that Block IL-36α and γ Signaling: Fusculoma cells seeded in 15-well microplates formed multiple colonies within the wells and reached an appropriate size. The aim was to screen for fusculomas using the binding activity of the anti-IL-36R antibody against human IL-36R as an indicator. Cell ELISA was performed using cells stably expressing human IL-36R. CHO cells stably expressing human IL-36R were prepared at a concentration of 1×10⁶ cells / mL and seeded at 100 μL / well in a poly-D-lysine 96-well plate (Corning, 356461). After one night of culture, the culture medium was discarded, and fusculoma culture supernatant was added to a concentration of 30 μL / well. The reaction was carried out at room temperature for 1 hour.

[0117] After reacting the fusion tumor culture supernatant, discard the reaction solution containing anti-IL-36R antibody and wash twice with PBS containing 0.05% Tween 20 (PBS-T). After washing, discard the PBS-T and fix the cells with 10% neutral buffered formalin solution at room temperature for about 10 minutes. After cell fixation, discard the 10% neutral buffered formalin solution, wash once with PBS-T, and add 30 μL / well of anti-rat IgG antibody HRP (Horse Radish Peroxidase) conjugation solution (SouthernBiotech, 3030-05) diluted 5000 times with 0.2% FBS / 3% BSA / PBS, and react at room temperature for about 1 hour. After the secondary antibody reaction, the samples were washed four times with PBS-T. Then, 100 μL / well of the chromogenic matrix TMB (3,3',5,5'-tetramethylbenzidine) (Thermofisher Sceintific, 34021) solution, prepared according to the manufacturer's experimental guidelines, was added to initiate the reaction. While observing the degree of color development, 100 μL / well of 2N sulfuric acid was added after approximately 20 minutes to stop the reaction. The absorbance was measured at 450 nm and 650 nm, and the difference between (absorbance at 450 nm) and (absorbance at 650 nm) was calculated as the OD value (absorbance). The absorbance values ​​were analyzed and statistically analyzed (mean, standard deviation, maximum, and minimum were calculated). For two-stage screening, an absorbance of 0.3 was set as the threshold for selecting superior strains, and the culture supernatant of 458 superior strains was recovered. For 458 colonies, using 80 colonies / microplate, the inhibitory activity of the fusion tumor supernatant was evaluated by the following analysis.

[0118] Using the cells for functional evaluation described in Example 3, cells were seeded at 1×10⁴ cells / well and cultured overnight. The following day, after removing the culture medium, only 30 μL of culture medium was added to the wells used in the control group, and 30 μL of culture supernatant of the fusion tumor was added to the wells used in the evaluation group.

[0119] After 30 minutes, add 30 μL / well of active human IL-36α (6995-IL / CF, manufactured by R&D Company) prepared in medium at a concentration of 300 ng / mL. For the negative control group, add medium to the wells containing the added medium; for the positive control group, add medium containing IL-36α ligand to the wells containing the added medium. Incubate at 37°C and 5% CO2 for 5 hours. Remove the medium containing the ligand solution, and add 100 μL / well of Dual-Glo (registered trademark) fluorescent enzyme assay reagent diluted twice with PBS. Gently shake for 10 minutes in the dark, and measure using a Tristar LB941 (Berthold Technologies) under the conditions of Counting Time: 0.1 sec, Emission Filter: No filter slot-A8. The measured value is the F-value. The F value of any well is labeled Fx, the average F value at the maximum ligand concentration is labeled Fposi, and the average F value of the negative control group is labeled Fnega.

[0120] Next, 50 μL / well was added to diluted Dual-Glo (registered trademark) Stop & Glo (registered trademark) reagent, and the mixture was slowly shaken for 10 minutes under light-protected conditions. The determination was then performed under the same conditions. The measured value was recorded as the R value. The R value of any well was labeled Rx, the average R value of the highest ligand concentration was labeled Rposi, and the average R value of the negative control group was labeled Rnega.

[0121] When the average value of the information from the pores in the positive control group is 100%, the NFkB information of each ligand of IL-36α, β or γ in any pore x is defined as follows.

[0122] Figure 6 shows the relationship between the binding screening results induced by anti-IL-36R antibody and the IL-36α signaling inhibition screening results. After comparing and evaluating the binding activity (absorbance) and IL-36α signaling inhibition activity of the culture supernatant of the 458 fusion tumors evaluated, there was no correlation between binding strength and inhibition activity.

[0123] Next, using the aforementioned evaluation method, the culture supernatant of fusion tumors generated by 111 antibodies that made the NFkB signal (%) of IL-36α less than 60% and the culture supernatant of fusion tumors generated by 3 antibodies with no blocking activity were evaluated. The blocking activity of the culture supernatant of fusion tumors on IL-36α, IL-36β, and IL-36γ at a final concentration of 150 ng / mL was evaluated. The IL-36β NFkB signal (%) or IL-36γ NFkB signal (%) was plotted against the IL-36α NFkB signal (%) to analyze the relative signal intensity.

[0124] The results of IL-36R signal blocking (NFkB Signal (%)) of the selected anti-IL-36R antibodies, showing the effects of IL-36β or IL-36γ signal blocking relative to IL-36α signal blocking, are shown in Figures 7A and 7B, respectively. From these results, the signal blocking activities of IL-36α and IL-36γ show the same tendency; in other words, when the IL-36α NFkB Signal (%) is equal to the IL-36β NFkB Signal (%) or IL-36γ NFkB Signal (%), the data are plotted on the straight line. On the other hand, the existence of antibodies with relatively weak IL-36β signal blocking activity was confirmed. From the results of this study, it is known that there are fusion tumor culture supernatants that block IL-36R signaling of IL-36α and IL-36γ, but do not block IL-36β's IL-36R signaling.

[0125] [Example 7] Screening of anti-human IL-36R antibodies that do not block IL-36β messages. The second cell fusion was performed using the method described in Example 5. Fusion cells (fusion tumors) seeded in 10-well discs formed multiple colonies in the wells and reached an appropriate size. The culture supernatant of the fusion tumors used as the first screening was evaluated for its inhibitory activity against stimulation with a final concentration of 150 ng / mL of IL-36α and IL-36β using the method described in Example 6.

[0126] The signal intensity of IL-36α and IL-36β was evaluated in the culture supernatant of the fusion tumor. A graph was plotted with the IL-36α NFkB Signal (%) of the well containing the culture supernatant as the x-axis and the IL-36β NFkB Signal (%) as the y-axis. The results are shown in Figure 8. Based on these results, strains in which no IL-36β signal inhibition was observed under these screening conditions were identified (strains represented by the circles indicated by the dashed lines in the figure).

[0127] [Example 8] Determination of amino acid sequence, isotyping, and antibody preparation of anti-IL-36R antibody: Fusible tumors (CL_00071: fusion tumor of test example 1) contained in positive wells that specifically inhibit the activity of anti-IL-36R antibodies, and fusion tumors (CL_00055 and CL_00069: fusion tumors of reference examples 1 and 2) that produced anti-hIL-36R antibodies that similarly inhibited all IL-36R messages, were monocultured using the limiting dilution method. Whole RNA was extracted from cell clumps of fusion tumors from each test example and reference example using an RNA extraction kit (Qiagen, 74106) following the method described in the product appendix. After quantifying the RNA concentration by absorbance, cDNA was prepared from 1 μg of whole RNA using an Omniscript RT Kit (Qiagen, 205113) following the method described in the product appendix. The antibody gene in the fusion tumor strain was determined by PCR using the primer described in International Patent Publication No. 2006 / 106366 (Patent Document 5). Using the prepared cDNA as a template, the gene encoding the variable region of the antibody was amplified by a PCR reaction employing DNA polymerase. The PCR reaction consisted of 50 μL of reaction solution containing the prepared cDNA, primer set (final concentration 1 μM each), and DNA polymerase, repeated 30 times at 98°C for 10 seconds, 55°C for 10 seconds, and 68°C for 30 seconds.

[0128] When no PCR amplification of the gene is observed using the primer set used, or when the obtained antibody gene is inactive, the primer is remade based on the gene information encoding the signal peptide of the antibody registered in IMGT (Immuno Gene Tics: http: / / www.imgt.org / ) for the same investigation, or the base sequence of the variable region of the antibody is determined by the 5' RACE method.

[0129] Antibody purification and isotype confirmation were performed as follows. 10-15 mL of culture medium was added to appropriately sized culture dishes to culture fusion nodules. The resulting culture supernatant was centrifuged at 15000 g for 10 minutes to clarify the supernatant. The antibody was purified using a batch method with protein G. The protein G resin was recovered into an open column, washed with sufficient buffer, and then dissolved in 0.1 M glycine solution at pH 2.8. The solution was immediately neutralized with 1 M Tris-HCl at pH 8. Several fractions were obtained. For fractions that could be confirmed to an absorbance of 280 nm using a Nanodorop 2000 (Thermofisher Sceintificate), the dissolved solution was concentrated using a centrifuge membrane concentrator with a molecular weight cutoff of 30,000 Da. Add at least 10 times the volume of PBS to the concentrate, and perform the concentration process at least three more times. Replace PBS with PBS, and prepare a 1 mg / mL solution with an absorbance (optical path length 10 mm) of 1.37. (Alternatively, antibodies purified from the culture supernatant of fusion tumors are described as antibodies purified from fusion tumor supernatant.) Evaluations were conducted using the PBS-replaced solution. Antibody isotype was determined using a rat antibody isotype identification kit (ANP: Antagen Pharmaceuticals, ISO-R8-20). All molecular weights of the antibodies were calculated in 150 kDa. The antibodies generated from the fusion tumors of each test case and reference case (antibodies of test case 1 and reference cases 1 and 2) were codon-optimized gene sequences artificially synthesized, encoding the amino acid sequences of the variable region of the antibody and the constant region consistent with the results of isotype identification. These sequences were inserted into pcDNA3.4 (Thermofisher Scientific, A14697) or pCAG-Neo (Wako Pure Chemicals, 163-25601). The plasmonic system was purified using an endotoxin-free plasmonic preparation kit (MACHEREY-NAGEL, 740422.50). The recombinant antibody system was obtained using a transient expression system for the heavy and light chains of the antibody introduced into planktonic CHO cells (Thermofisher Sceintific, A29133). After appropriate culture, when the antibodies necessary for evaluation were obtained, the culture supernatant was recovered and purified in the same manner as the antibodies derived from the fusion tumors. Furthermore, when purifying antibodies with human constant regions, the resin used for purification is changed to Protein A, and the dissolution solution used for dissolution is changed to a 0.1M sodium acetate aqueous solution with pH 3.3. The same purification process is performed as for antibodies with rat constant regions.

[0130] The amino acid sequences of the variable regions of the heavy and light chains of the anti-IL-36R antibody obtained in Test Example 1 are shown in Figure 9. Furthermore, the selection results of the anti-IL-36R antibodies from Reference Examples 1 and 2 and Test Example 1 are shown in the table below. The "Recombinant / Fusion Tumor Name" column in the table shows the name of the recombinant antibody and the corresponding name of the antibody-generated fusion tumor. The "Antibody Function" column in the table shows the selectivity of the antibody's inhibition against each IL-36 stimulus.

[0131]

[0132] Furthermore, based on the amino acid sequence of the antibody obtained in Example 1, and using the germline sequence as a reference, mutant antibodies (antibodies of Examples 2-9) were formulated to specifically inhibit the introduction of IL-36R signaling mutations into the amino acid sequence. Similar to the selection results of the antibodies in Reference Examples 1 and 2 and Example 1, the sequence information of the antibodies in Examples 2-9, 117hG1Dk, and 117hG4k is shown in the table below. 117hG1Dk and 117hG4k are chimeric antibodies possessing the variable region of the antibody in Example 1 and the constant region of the human antibody. Furthermore, the "Recombinant Name" column in the table displays the name of the recombinant antibody.

[0133]

[0134] [Example 9] Evaluation of the binding activity of IL-36R-specific inhibitory antibody against human IL-36R (Cell ELISA) (9-1) Evaluation of antibody purified from fusion tumor supernatant. To investigate the binding activity of anti-IL-36R antibody derived from the culture supernatant of fusion tumors from Reference Examples 1 and 2 and Experimental Example 1 against human IL-36R, Cell ELISA was performed using cells stably expressing human IL-36R. CHO cells stably expressing human IL-36R were seeded at 1×10⁵ cells / well in poly-D-lysine 96-well plates (Corning, 356461). After one night of culture, the culture medium was discarded, and serial dilutions of anti-IL-36R antibody and rat isotype control group (R&D, MAB0061) were added to a final volume of 30 μL / well. The reaction was carried out at room temperature for 1 hour. Furthermore, the anti-IL-36R antibody dilution series consisted of adjusting the anti-IL-36R antibody solution to 15 μg / mL with culture medium, followed by 11 stages of 3-fold dilution with culture medium. The rat isotype control group dilution series consisted of preparing the solution to 15 μg / mL with culture medium, followed by 5 stages of 9-fold dilution with culture medium. After reacting the anti-IL-36R antibody and isotype control group dilutions for approximately 1 hour, the reaction solution containing either the anti-IL-36R antibody or the isotype control group was discarded, and the cells were washed twice with PBS containing 0.05% Tween 20 (PBS-T). After washing, the PBS-T was discarded, and the cells were fixed with 10% neutral buffered formalin at room temperature for approximately 10 minutes. Then, the 10% neutral buffered formalin was discarded, and the cells were washed once with PBS-T. After washing, discard PBS-T and add 30 μL / well of anti-rat IgG antibody HRP conjugation solution diluted 5000 times with 0.2% FBS / 3% BSA / PBS. Incubate at room temperature for approximately 1 hour. Discard the anti-rat IgG antibody HRP conjugation solution and wash four times with PBS-T. After washing, discard PBS-T and add 100 μL / well of TMB solution (Thermofisher Sceintificate, 34021) prepared according to the manufacturer's experimental instructions to start the reaction. After approximately 20 minutes, add 2N sulfuric acid to make 100 μL / well and stop the reaction. Measure the absorbance at 450 nm and 650 nm using a microdisc reader and calculate (absorbance at 450 nm) - (absorbance at 650 nm) as the OD value. Using GraphPad Prism (registered trademark) Ver8.2.1, the dosage-response curve was fitted with log(agonist) vs. response-variable slope (four parameters) to calculate the EC50 value. The results are shown in the table below.

[0135]

[0136] (9-2) Evaluation using recombinant antibodies: To investigate the binding activity of recombinant anti-IL-36R antibody to human IL-36R, Cell ELISA was performed using cells stably expressing human IL-36R. Stable human IL-36R-expressing CHO cells were seeded at 1×10⁵ cells / well in poly-D-lysine 96-well plates (Corning, 356461). After one night of culture, the culture medium was discarded, and 30 μL / well of recombinant anti-IL-36R antibody serial dilution and rat isotype control group serial dilution were added, and the reaction was carried out at room temperature for 1 hour. Furthermore, the recombinant anti-IL-36R antibody serial dilution was prepared with culture medium to a concentration of 15 μg / mL, and then diluted 3-fold in 11 stages with culture medium. Similarly, the rat isotype control group serial dilution was prepared with culture medium to a concentration of 15 μg / mL, and then diluted 9-fold in 5 stages with culture medium. After reacting the anti-IL-36R antibody solution and the rat isotype control group solution for approximately 1 hour, discard the reaction solution containing the anti-IL-36R antibody or the rat isotype control group, and wash twice with PBS containing 0.05% Tween 20 (PBS-T). After washing with PBS-T, fix the cells with 10% neutral buffered formalin solution at room temperature for approximately 10 minutes. Discard the 10% neutral buffered formalin solution and wash once with PBS-T. Discard the PBS-T, and add 30 μL / well of anti-rat IgG antibody HRP conjugation solution diluted 5000 times with 0.2% FBS / 3% BSA / PBS, and react at room temperature for approximately 1 hour. Discard the anti-rat IgG antibody HRP conjugation solution and add PBS-T. Repeat the steps of discarding PBS-T and adding PBS-T 4 times. Discard PBS-T and add TMB solution (Thermofisher Sceintificate, 34021) prepared according to the manufacturer's experimental guidelines to a final volume of 100 μL / well to begin the reaction. After approximately 20 minutes, add 2N sulfuric acid to a final volume of 100 μL / well to stop the reaction. Measure the absorbance at 450 nm and 650 nm using a microdisk absorbance reader, and calculate the difference between (absorbance at 450 nm) and (absorbance at 650 nm) as the OD value. Use GraphPad Prism (registered trademark) Ver8.2.1 to fit the dosage-response curve using log (agonist) vs. response-variable slope (four parameters) to calculate the EC50 value. The results are shown in the table below.

[0137]

[0138] [Example 10] Analysis of the binding site of IL-36R message-specific blocking antibody (flow cytometry, Cell ELISA), domain recombination. In order to identify the binding domain of anti-IL-36R antibodies purified from the culture supernatant of fusion tumors of Reference Examples 1 and 2 and Test Example 1 to human IL-36R (hIL-36R), the binding of anti-IL-36R antibodies to IL-36R domain recombinant variants in which the domain of human IL-36R was replaced with the domain of mouse IL-36R was measured.

[0139] Specifically, in the amino acid sequence of human IL-36R shown in sequence number 11, region X1-Y1 (amino acid residues 1-100) is designated as extracellular domain I, region X2-Y2 (amino acid residues 101-198) is designated as extracellular domain II, and region X3-Y3 (amino acid residues 199-316) is designated as extracellular domain III. The following 6 IL-36R variants (or alternatively referred to as "substitutes") are created by substituting one or two of the three domain sequences with the domains corresponding to mouse IL-36R.

[0140] (Variant 1: m1h2h3) A variant in which the sequence of domain I of human IL-36R is replaced with the sequence of domain I of mouse IL-36R. (Variant 2: h1m2h3) A variant in which the sequence of domain II of human IL-36R is replaced with the sequence of domain II of mouse IL-36R. (Variant 3: h1h2m3) A variant in which the sequence of domain III of human IL-36R is replaced with the sequence of domain III of mouse IL-36R. (Variant 4: m1m2h3) A variant in which the sequences of domain I and domain II of human IL-36R are replaced with the sequences of domain I and domain II of mouse IL-36R. (Variant 5: m1h2m3) A variant in which the sequences of domain I and domain III of human IL-36R are replaced with the sequences of domain I and domain III of mouse IL-36R. (Variant 6: h1m2m3) is a variant in which the domain II and domain III sequences of human IL-36R are replaced with the domain II and domain III sequences of mouse IL-36R.

[0141] The amino acid sequences of the obtained IL-36R domain recombination variants 1 to 6 and the sequence numbers encoding these base sequences are shown in the table below.

[0142]

[0143] HEK293 cells were transfected with pCAG-Neo vectors containing genes encoding the amino acid sequences of the six IL-36R variants 1-6 (sequence numbers 90, 92, 94, 96, 98, and 100) via lipid transfection and cultured overnight. Various anti-hIL-36R antibodies were diluted to 5 μg / mL with cell staining buffer. After culture, the gene-transfected HEK293 cells were recovered from the culture dish, and the diluted anti-hIL-36R antibodies were incubated at room temperature for 20 minutes. After washing once with cell staining buffer, the cells were reacted with a 1000-fold diluted anti-rat IgG-Alexa Fluor (registered trademark) 488 antibody (Thermo Fisher Scientific, A-11006) as a secondary antibody for 20 minutes. After washing once, the cells were resuspended at an appropriate concentration and analyzed by flow cytometry using an Accuri C6 plus (Becton Dickinson) instrument. Furthermore, negative-cotrol aims to prevent primary antibody responses, such as those against IL-36R, from occurring on cells exhibiting various variants, instead eliciting only secondary antibody responses. After gated by FSC and SSC, a threshold is set with the number of positive cells reaching 0%. Based on this threshold, antibody binding to each variant is expressed as the positive cell percentage (the proportion of cells exceeding the threshold relative to the total cell count). The results are shown in the table below. A positive cell percentage below 5% is considered non-binding and is indicated by "-" in the table. Conversely, a positive cell percentage above 20% is considered binding and is indicated by "+" in the table. For percentages above 5% but below 20%, the values ​​are indicated by "±".

[0144]

[0145] Furthermore, the binding domain was confirmed by Cell ELISA. HEK293 cells were transfected with pCAG-Neo vectors containing the amino acid sequences encoding the above-mentioned six human IL-36R variants 1-6 (sequence numbers 90, 92, 94, 96, 98, and 100) via lipid transfection and cultured overnight. Cells were recovered from the culture dish and seeded at 1×10⁵ cells / well in a poly-D-lysine 96-well plate (Corning, 356461). After one night of culture, the culture medium was discarded, and serial dilutions of anti-IL-36R antibody and rat isotype control group (R&D, MAB0061) were added to a concentration of 30 μL / well, and the reaction was carried out at room temperature for 1 hour. Furthermore, the anti-IL-36R antibody and rat isotype control group dilutions were prepared using culture medium (10% FBS, penicillin / streptomycin solution / DMEM) adjusted to 3.75 μg / mL, and then diluted to 1.5 μg / mL, 0.75 μg / mL, 0.15 μg / mL, 0.03 μg / mL, and 0.006 μg / mL. After reacting the antibody dilutions for approximately 1 hour, the reaction solutions containing anti-IL-36R antibody or rat isotype control group antibody were discarded, and the cells were washed twice with PBS containing 0.05% Tween 20 (PBS-T). After washing, the PBS-T was discarded, and the cells were fixed with 10% neutral buffered formalin at room temperature for approximately 10 minutes. The 10% neutral buffered formalin was discarded, and the cells were washed once with PBS-T. After washing, discard PBS-T and add 30 μL / well of anti-rat IgG antibody HRP conjugation solution (SouthernBiotech, 3030-05) diluted 5000 times with 0.2% FBS / 3% BSA / PBS. Incubate at room temperature for approximately 1 hour. Discard the anti-rat IgG antibody HRP conjugation solution and add PBS-T. Discard PBS-T and wash 4 times with PBS-T. After washing, discard PBS-T and add 100 μL / well of TMB solution (Thermofisher Sceintific, 34021) prepared according to the manufacturer's instructions. Begin the reaction. After approximately 20 minutes, add 2N sulfuric acid to make 100 μL / well and stop the reaction. The absorbance at 450 nm and 650 nm was measured using a SpectraMax 190 (Molecular Devices) microdisk reader. The difference between (absorbance at 450 nm) and (absorbance at 650 nm) was calculated as the OD value (absorbance). The dosage-response curve was fitted using GraphPad Prism (registered trademark) Ver8.2.1 with log(agonist) vs. response-variable slope (four parameters), and the result was plotted.

[0146] The results are shown in Figure 10 and the table below. At a concentration of 3.75 μg / mL, absorbance of 0.9 or higher is considered bound, and is indicated as "+" in the table below. Conversely, absorbance below 0.5 indicates a significant decrease in binding, and is indicated as "-" in the table below. Absorbance between 0.9 and 0.5 is indicated as "±".

[0147]

[0148] Based on the above results, the antibody of Test Example 1 binds to all receptors containing domain I (h1) of human IL-36R, but does not bind to any receptors containing domain I (m1) of mouse IL-36R. On the other hand, it can be seen that the antibodies of Reference Examples 1 and 2 bind to receptors containing domain II (h2) of human IL-36R. From these results, it is confirmed that the antibody system of Test Example 1 uses domain I as the antigenic determinant.

[0149] [Example 11] Determination of the antigenic determinant of anti-hIL-36R antibody (Cell ELISA) Since the IL-36R signal-specific blocking antibody in Example 1 is an antibody that binds to domain I, in order to limit the binding site, a variant was prepared by substituting a portion of the amino acid residues from the N-terminus of IL-36R sequence number 11, from amino acid residues 1 to 112, in an order of 8 residues each time, to form the sequence of mouse IL-36R. Another variant of IL-36R was prepared by substituting a portion of the amino acid residues from the N-terminus, from amino acid residues 5 to 108, in an order of 8 residues each time, to form the sequence of mouse IL-36R. Specifically, the following 25 IL-36R variants were prepared.

[0150] (Mutant 7) Mutants obtained by replacing glycine in No. 2 with threonine and lysine in No. 4 with glutamic acid: G2T, K4E. (Mutant 8) Mutants obtained by replacing lysine in No. 9 with histamine and glutamic acid in No. 11 with valine: K9H, E11V. (Mutant 9) Mutants obtained by replacing lysine in No. 9 with histamine, glutamic acid in No. 11 with valine, leucine in No. 13 with isoleucine, alanine in No. 15 with glutamic acid, and serine in No. 16 with glycine: K9H, E11V, L13I, A15E, S16G. (Mutant 10) Variants obtained by replacing leucine in part 13 with isoleucine, alanine in part 15 with glutamic acid, serine in part 16 with glycine, and alanine in part 20 with proline: L13I, A15E, S16G, A20P. (Mutant 11) Variants obtained by replacing phenylalanine in part 25 with tyrosine and isoleucine in part 28 with glutamic acid: F25Y, I28E. (Mutant 12) Variants obtained by replacing phenylalanine in part 25 with tyrosine, isoleucine in part 28 with glutamic acid, serine in part 30 with asparagine, and glutamic acid in part 32 with alanine: F25Y, I28E, S30N, E32A. (Mutant 13) Variants obtained by replacing serine (number 30) with asparagine, glutamic acid (number 32) with alanine, serine (number 34) with asparagine, and valine (number 35) with leucine: S30N, E32A, S34N, V35L. (Mutant 14) Variants obtained by replacing serine (number 34) with asparagine, valine (number 35) with leucine, and asparagine (number 40) with threonine: S34N, V35L, N40T. (Mutant 15) The following mutants were obtained by replacing asparagine (No. 40) with threonine, serine (No. 41) with proline, and isoleucine (No. 44) with serine: N40T, S41P, I44S. (Mutant 16) The following mutants were obtained by replacing serine (No. 41) with proline, isoleucine (No. 44) with serine, and lysine (No. 48) with asparagine: S41P, I44S, K48N. (Variant 17) Variants obtained by replacing lysine in No. 48 with asparagine, isoleucine in No. 49 with asparagine, isoleucine in No. 50 with arginine, glutamine in No. 51 with histamine, and serine in No. 52 with leucine: K48N, I49N, I50R, Q51H, S52L.(Mutant 18) Variants obtained by replacing isoleucine (No. 49) with asparagine, isoleucine (No. 50) with arginine, glutamine (No. 51) with histidine, serine (No. 52) with leucine, and isoleucine (No. 54) with valine: I49N, I50R, Q51H, S52L, I54V. (Mutant 19) Variants obtained by replacing isoleucine (No. 54) with valine and glutamine (No. 58) with glutamine: I54V, E58Q. (Mutant 20) Variant obtained by replacing glutamine (No. 58) with glutamine: E58Q. (Variant 21) Variants obtained by replacing methionine (No. 66) with leucine, glutamic acid (No. 67) with threonine, and tryptophan (No. 68) with leucine: M66L, E67T, W68L. (Variant 22) Variants obtained by replacing methionine (No. 66) with leucine, glutamic acid (No. 67) with threonine, tryptophan (No. 68) with leucine, and glycine (No. 69) with glutamic acid: M66L, E67T, W68L, G69E. (Variant 23) Variants obtained by replacing glycine (No. 69) with glutamic acid and valine (No. 73) with isoleucine: G69E, V73I. (Variant 24) Variant obtained by replacing valine in No. 73 with isoleucine, lysine in No. 79 with arginine, and glycine in No. 80 with asparagine: V73I, K79R, G80N. (Variant 25) Variant obtained by replacing lysine in No. 79 with arginine, glycine in No. 80 with asparagine, arginine in No. 81 with alanine, asparagine in No. 82 with histidine, and serine in No. 83 with asparagine: K79R, G80N, R81A, D82H, S83N. (Variant 26) Variant 81, which replaces arginine with alanine, aspartic acid with histamine with aspartic acid with aspartic acid with aspartic acid with serine ... (Variant 28) Variants obtained by replacing phenylalanine in No. 94 with leucine, glutamic acid in No. 95 with lysine, and lysine in No. 96 with asparagine: F94L, E95K, K96N.(Variant 29) Variants obtained by replacing threonine in No. 101 with serine, isoleucine in No. 103 with methionine, and glycine in No. 104 with glutamic acid: T101S, I103M, G104E. (Variant 30) Variants obtained by replacing threonine in No. 101 with serine, isoleucine in No. 103 with methionine, glycine in No. 104 with glutamic acid, leucine in No. 106 with serine, and asparagine in No. 108 with valine: T101S, I103M, G104E, L106S, N108V. (Variant 31) A variant obtained by replacing leucine in No. 106 with serine, replacing asparagine in No. 108 with valine, and replacing leucine in No. 109 with asparagine, and a variant obtained by inserting proline in No. 111: L106S, N108V, L109N, 111P.

[0151] The amino acid sequences of the above IL-36R variants 7~31 and the sequence numbers encoding their base sequences are shown in the table below.

[0152]

[0153] pCAG-Neo vectors containing the genes of the aforementioned 25 IL-36R variants were transfected into HEK293 cells via lipid transfection and cultured overnight. HEK293 cells were then recovered from the culture dish and seeded at 1×10⁵ cells / well in a poly-D-lysine 96-well plate (Corning, 356461). After one night of culture, the culture medium was discarded, and serial dilutions of anti-IL-36R antibody and rat isotype control group (R&D, MAB0061) were added to a final volume of 30 μL / well. The mixture was incubated at room temperature for 1 hour. Furthermore, the anti-IL-36R antibody and rat isotype control group dilutions were prepared by adjusting the anti-IL-36R antibody solution or rat isotype control group to 3.75 μg / mL with culture medium (10% FBS, penicillin / streptomycin solution / DMEM), and then diluting them to 1.5 μg / mL, 0.75 μg / mL, 0.15 μg / mL, 0.03 μg / mL, and 0.006 μg / mL with culture medium. After reacting the anti-IL-36R antibody solution and isotype control group solution for about 1 hour, the reaction solution containing anti-IL-36R antibody or isotype control group was discarded, and the cells were washed twice with PBS containing 0.05% Tween 20 (PBS-T). After washing, the PBS-T was discarded, and the cells were fixed with 10% neutral buffered formalin solution at room temperature for about 10 minutes. The 10% neutral buffered formalin solution was discarded, and the cells were washed once with PBS-T. After washing, discard PBS-T and add 30 μL / well of anti-rat IgG antibody HRP conjugation solution (SouthernBiotech, 3030-05) diluted 5000 times with 0.2% FBS / 3% BSA / PBS. Incubate at room temperature for approximately 1 hour. Discard the anti-rat IgG antibody HRP conjugation solution and wash four times with PBS-T. After washing, discard PBS-T and add 100 μL / well of TMB solution (Thermofisher Sceintific, 34021) prepared according to the manufacturer's experimental instructions. Start the reaction. After approximately 20 minutes, add 2N sulfuric acid to make 100 μL / well and stop the reaction. Measure the absorbance at 450 nm and 650 nm, and calculate (absorbance at 450 nm) - (absorbance at 650 nm) as the OD value (absorbance).

[0154] The results are shown in the table below. In this experiment, the performance of each variant was confirmed by flow cytometry after temporary gene introduction. A decrease of more than 40% in performance compared to IL-36R was considered unsuitable for evaluation, and is indicated as "NA" in the table below. Based on the binding amount (OD value) of human IL-36R at an antibody concentration of 5 nM (0.75 μg / mL), the ratio of binding amount (OD value) of each variant in Experiment 1 was calculated (relative binding amount: refer to the formula below). A relative binding amount of 80% or more was considered as no change (indicated as "-" in the table), a relative binding amount of 50% or more but less than 80% was considered as a decrease (indicated as "±" in the table), and a relative binding amount less than 50% was considered as a significant decrease (indicated as "+" in the table). Antigenic determinants were determined by "+" in the table.

[0155]

[0156] The antibody in Example 1 did not bind to variant 7, thus suggesting the possibility that glycine (amino acid sequence 2) and lysine (amino acid sequence 4) of the IL-36R amino acid sequence are antigenic determinants. Furthermore, the antibody in Example 1 did not bind to variants 11 and 12, thus suggesting the possibility that phenylalanine (amino acid sequence 25) and isoleucine (amino acid sequence 28) are antigenic determinants. On the other hand, the antibody in Example 1 bound to variant 8 but not to variant 9. Also, the antibody in Example 1 bound to variants 25 and 27, but not to variant 26.

[0157] As shown in the structure of IL-36R disclosed in non-patent literature or in Figure 3, the variant site of variant 26 is structurally similar to that of variant 7 and variant 11 or 12. Although the binding of the antibody to variant 26 is different from that of variant 25 and variant 27, the structural analysis indicates that the site is contained within the antigen determinant.

[0158] [Example 12] Determination of antigenic determinants of anti-hIL-36R antibody (Cell ELISA) Based on the results of the investigation to date, it is suggested that glycine No. 2, lysine No. 4, phenylalanine No. 25, and isoleucine No. 28 in the amino acid sequence (sequence number 11) of hIL-36R are important for the specific inhibition of antibody binding to IL-36R information in Experimental Example 1. Therefore, various alanine variants of hIL-36R were created through the introduction of mutations (G2A (sequence number 151), which replaces glycine at position 2 of sequence 11 with alanine; K4A (sequence number 153), which replaces lysine at position 4 with alanine; F25A (sequence number 155), which replaces phenylalanine at position 25 with alanine; and I28A (sequence number 157), which replaces isoleucine at position 28 with alanine)). These were named variants 32, 33, 34, and 35, respectively. The genes for the artificially synthesized G2A variant (sequence number 152), K4A variant (sequence number 154), F25A variant (sequence number 156), and I28A variant (sequence number 158) were also developed.

[0159] HEK293 cells were transfected with pCAG-Neo vectors containing genes for the four IL-36R amino acid variants mentioned above using lipid transfection and cultured for one night. HEK293 cells were recovered from the culture dish and seeded at 1×10⁵ cells / well in a poly-D-lysine 96-well plate. After one night of culture, the culture medium was discarded, and serial dilutions of anti-IL-36R antibody and rat isotype control group (R&D, MAB0061) were added to make 30 μL / well, and the reaction was carried out at room temperature for 1 hour. Furthermore, the anti-IL-36R antibody and rat isotype control group dilutions were prepared by adjusting the anti-IL-36R antibody or rat isotype control group solution to 3.75 μg / mL with culture medium (10% FBS, penicillin / streptomycin solution / DMEM), and then diluting it to 1.5 μg / mL, 0.75 μg / mL, 0.15 μg / mL, 0.03 μg / mL, and 0.006 μg / mL with culture medium. After allowing the antibody solution to react for approximately 1 hour, the reaction solution containing the anti-IL-36R antibody or rat isotype control group was discarded, and the cells were washed twice with PBS containing 0.05% Tween 20 (PBS-T). After washing, the PBS-T was discarded, and the cells were fixed with 10% neutral buffered formalin solution at room temperature for approximately 10 minutes. The 10% neutral buffered formalin solution was discarded, and the cells were washed once with PBS-T. After washing, discard PBS-T and add 30 μL / well of anti-rat IgG antibody HRP conjugation solution (SouthernBiotech, 3030-05) diluted 5000 times with 0.2% FBS / 3% BSA / PBS. Incubate at room temperature for approximately 1 hour. Discard the anti-rat IgG antibody HRP conjugation solution and wash four times with PBS-T. After washing, repeat the steps of discarding and adding PBS-T four times. Discard PBS-T and add 100 μL / well of TMB solution (Thermofisher Sceintific, 34021) prepared according to the manufacturer's experimental instructions to start the reaction. After approximately 20 minutes, add 2N sulfuric acid to make 100 μL / well and stop the reaction. Measure the absorbance at 450 nm and 650 nm, and calculate (absorbance at 450 nm) - (absorbance at 650 nm) as the OD value (absorbance).

[0160] The anti-IL-36R antibody was prepared using the antibodies from Test Examples 1 and 2, as well as Reference Examples 1 and 2. The reference antibody system was selected based on the EC50 and Emax of the antibody in Test Example 1, which were close to those of the antibody in Test Example 1. Furthermore, the antibody in Test Example 1 was prepared using both purified antibody and recombinant antibody obtained from fusion tumor supernatant. The absorbance ratios of the antibodies in Test Examples 1 and 2, and Reference Example 2, relative to the antibody in Reference Example 1, were calculated at an antibody concentration of 0.75 μg / mL.

[0161] The results are shown in the table below. When the absorbance of Reference Example 1 is 100% and the relative binding amount is 90% or more, it is judged as the possibility of no antigenic determinant (marked as "-" in the table); when the relative binding amount is 70% or more but less than 90%, it is judged as the possibility of having a few antigenic determinants (marked as "±" in the table); when the relative binding amount is less than 70% but more than 50%, it is judged as an antigenic determinant (marked as "+" in the table); when the relative binding amount is less than 50%, it can be considered as having a strong correlation and is judged as a particularly important antigenic determinant (marked as "++" in the table).

[0162]

[0163] The results of this study show that the fourth lysine (replaced with Ala in variant 33) and the 28th isoleucine (replaced with Ala in variant 35) from the N-terminus of human IL-36R (sequence number 11) are important antigenic determinants of Experimental Example 1.

[0164] [Example 13] Evaluation of IL-36R signaling inhibition using reporter gene analysis of IL-36R signaling-specific inhibitory antibodies (13-1) Evaluation of antibodies purified from fusion tumor supernatant using the method described in Example 6, by stimulating cells with IL-36α at a final concentration of 50 ng / mL, IL-36β at a final concentration of 10 ng / mL, and IL-36γ at a final concentration of 50 ng / mL, respectively, to evaluate the inhibitory activity of the antibody. The dilutions of anti-human IL-36R antibody and rat isotype control group were based on a final concentration of 100 nM and the highest concentration, and were used after 7 stages of 4-fold dilution. The NFkB signal% of each well was calculated using Equation 2. The negative control group (0%) was set as data of the culture medium, and the positive control group (100%) was set as data of each IL-36 active agent. The dosage-response curve was fitted using GraphPad Prism (registered trademark) Ver8.2.1 with log(inhibitor) vs. response - variable slope (four parameters). From the regression analysis results, the antibody concentration with Y=50 was calculated using the formula: Y=Bottom +(Top-Bottom) / (1+10^((LogIC50-X)*HillSlope)), which was taken as the 50% inhibition concentration (IC50). The antibody system used was purified from the supernatant of the fusion tumors in Experimental Example 1 and Reference Examples 1 and 2.

[0165] The results are shown in Figure 11 and the table below. Antibodies derived from all fusion tumor strains showed inhibitory activity against IL-36α and IL-36γ, but the antibody in Test Example 1 did not show inhibitory activity against IL-36β. Furthermore, in the table below, the highest concentration of antibody in the experiment is recorded as NA for antibodies whose inhibitory activity value did not reach 50%.

[0166]

[0167] (13-2) Evaluation of Recombinant Antibodies The recombinant antibodies were also evaluated using the same method as described above. The inhibitory activity of the antibodies against IL-36R signaling when cells were stimulated with IL-36α, IL-36β, and IL-36γ at a final concentration of 50 ng / mL was evaluated. The recombinant antibody system used the antibodies from Test Example 1 and Reference Example 1. IL-36Ra (maximum treatment concentration 17 μg / mL (equivalent to 1 μM)), an IL-36R antagonist, was also used for comparison. The results are shown in Figure 12 and the table below. The recombinant antibody from Test Example 1, similar to the antibody purified from the fusion tumor supernatant, did not inhibit IL-36β signaling. Antibodies whose inhibitory activity at the maximum treatment concentration did not reach 50% were recorded as NA.

[0168]

[0169] [Example 14] Evaluation of the inhibitory activity of IL-36β by an IL-36R signal-specific inhibitory antibody (recombinant antibody) (14-1) Evaluation of the effect of IL-36β concentration: Using the method described in Example 6, cells were stimulated with IL-36β at final concentrations of 2 ng / mL, 10 ng / mL, and 250 nmol / mL to evaluate the inhibitory activity of the antibody (n=2). The dilutions of the anti-human IL-36R antibody and the rat isotype control group were obtained by using a 4-fold dilution followed by 7 stages of dilution, as the highest concentration of 100 nM. The NFkB signal% of each well was calculated using Equation 2. The negative control group (0%) was set as the data of the culture medium, and the positive control group (100%) was set as the data of each IL-36 active agent. The dosage-response curve was fitted using GraphPad Prism (registered trademark) Ver8.2.1 with log (inhibitor) vs. response-variable slope (four parameters), and a graph was generated. The anti-inflammatory system used the recombinant antibodies from Test Example 1 and Reference Example 1. Additionally, IL-36Ra, an IL-36R antagonist, was used as a comparison. The results are shown in Figure 13. Regardless of the concentration of IL-36β, the antibody from Test Example 1 did not inhibit IL-36β in a concentration-dependent manner. Furthermore, the isotype control group did not inhibit any IL-36R agonist signal.

[0170] (14-2) Anti-IL-36R antibody was prepared in a series of dilutions (4-fold dilution, 10-stage dilution) with an antibody concentration exceeding 100 nM, using the highest treatment concentration as the final concentration of 1 μM. Cells were stimulated with IL-36α at a final concentration of 50 ng / mL, IL-36β at 10 ng / mL, and IL-36γ at 50 ng / mL, as described in Example 6, and the inhibitory activity of the antibody was evaluated (n=2). The NFkB signal% of each well was calculated using Equation 2. The negative control group (0%) was set as the data of the culture medium, and the positive control group (100%) was set as the data of each IL-36 agonist. The dosage-response curve was fitted using GraphPad Prism (registered trademark) Ver8.2.1 with log (inhibitor) vs. response-variable slope (four parameters), and plotted. The anti-antibody system used the recombinant antibodies of Test Example 1 and Reference Example 1.

[0171] The results are shown in Figure 14. The antibody in Experiment 1 did not block IL-36β signals even when the antibody concentration reached 1 μM.

[0172] [Example 15] Binding activity of IL-36R message-specific blocking antibody (recombinant antibody) against human IL-36R (Cell ELISA) The binding activity of the recombinant antibodies of Examples 2-6 and the recombinant antibodies against human IL-36R described in prior art documents (antibodies C81B4 and B3, C73C5 described in Patent Document 2, and antibodies APE3849 and APE6060 described in Patent Document 3) was evaluated using the method described in Example 9 (n=2). The dosage-response curve was fitted using GraphPadPrism (registered trademark) Ver8.2.1 with log (agonist) vs. response-variable slope (four parameters), and the EC50 value was calculated. The results are shown in the table below.

[0173]

[0174] [Example 16] Analysis of the binding domain and binding site of previous anti-IL-36R antibodies (16-1) used the method described in Example 10 to evaluate the binding of anti-IL-36R antibodies against human IL-36R described in previous art documents (antibodies described in Patent Document 2: C81B4, B3, and C73C5, and antibodies described in Patent Document 3: APE3849, APE6060, and APE7247). The binding of these antibodies was evaluated by flow cytometry. When the positive cell count was less than 5%, it was considered as no binding (-); when it was 5% or more but less than 20%, it was considered as ±; and when it was 20% or more, it was considered as binding (+). The results are shown in the table below.

[0175]

[0176] Based on the above results, it is confirmed that the anti-IL-36R antibodies in previous patent literature bind to recombinant variants of the IL-36R domain containing domain II of human IL-36R, rather than antibodies that bind to domain I of human IL-36R.

[0177] (16-2) Evaluation of IL-36R variants: Using human IL-36R variants 7, 26, 31, 11 and 8 as described in Example 11, the binding affinity of recombinant antibodies from Examples 2 to 6, rat / human chimeric antibodies (117hG1Dk and 117hG4k) prepared based on 117 (Example 1, C_00028) rat antibodies, and anti-IL-36R antibodies against human IL-36R described in prior art documents (antibodies C81B4 and C73C5 as described in Patent Document 2, and antibodies APE6060 and APE7247 as described in Patent Document 3) was evaluated using the method described in Example 11. A recombinant antibody with an absorbance of 0.9 or higher at a concentration of 3.75 μg / mL was considered to have bound antibodies and was indicated as "-" as a non-antigenic determinant. On the other hand, absorbance values ​​below 0.5 are considered to indicate a significant reduction in binding, which is identified as an antigenic determinant and indicated as "+".

[0178]

[0179] The recombinant antibodies of Test Examples 2-6, like those of Test Example 1, did not bind to variants 7, 11, and 26, but bound to variants 8 and 31. It is clear from this that the antigenic determinants of the recombinant antibodies of Test Examples 2-6 are the same as those of Test Example 1.

[0180]

[0181] The antibodies described in previous art literature bind to all the IL-36R variants investigated. Among those evaluated, there were no variants of antibodies described in previous art literature that showed reduced or no binding. On the other hand, the chimeric antibodies (117hG1Dk and 117hG4k) that changed the constant region of Test Example 1 to the human type, like the antibody in Test Example 1, did not bind to variants 7, 11, and 26, and the antigenic determinant was positive. Therefore, it is clear that the antibodies described in Test Examples 1-6 have different antigenic determinants than conventional anti-IL-36R antibodies.

[0182] [Example 17] The IL-36R signaling inhibition of the anti-IL-36R antibody using reporter gene analysis was assessed using the method described in Example 6. Cells were stimulated with IL-36α at a final concentration of 50 ng / mL, IL-36β at 10 ng / mL, and IL-36γ at 50 ng / mL to evaluate the inhibitory activity of the antibody. The dilutions of the anti-human IL-36R antibody and the rat isotype control group were obtained by 7 stages of dilution, with 100 nM as the maximum treatment concentration, and 4-fold dilution (NFkB signal% of each well was calculated by Equation 2). The dosage-response curve was fitted using GraphPad Prism (registered trademark) Ver8.2.1 with log (inhibitor) vs. response-variable slope (four parameters). Based on the regression analysis results, the antibody concentration at which Y=50 was calculated using the formula: Y=Bottom +(Top-Bottom) / (1+10^((LogIC50-X)*HillSlope)), which was taken as the 50% blocking concentration IC50. In addition to the antibody used in Example 1, Reference Example 1, Examples 2-9, and the anti-IL-36R antibodies B3, APE6060, and C73C5 described in previous technical literature were also used. The results are shown in the table below (Example 1 and the isotype control group are n=1 or n=2, and the others are n=2). For antibodies whose NFkB signal% did not reach 50% at the maximum blocking concentration (100nM, the highest antibody treatment concentration), the evaluation result was recorded as NA.

[0183]

[0184]

[0185]

[0186]

[0187] The isotype control group did not block all IL-36R agonist signals. The antibodies in Test Examples 1-9 blocked all IL-36α and IL-36γ signals, but unlike the antibodies in previous technical literature, they did not block IL-36β signals.

[0188] [Example 18] Evaluation of the stability of anti-IL-36R antibody. The sample used for evaluation (Example 1) was prepared with PBS (Thermofisher Scientific, 10010023) at a concentration of 1 mg / mL. The absorbance of the antibody at 280 nm (optical path length of 10 mm) was 1.49. 200 μL of the 1 mg / mL antibody was transferred to the following container and stored in a refrigerator (set to 4°C: Panasonic, MPR-1411R-PJ) or a cell culture device (set to 37°C: Panasonic, MCO-175-PJ) for 30 days (approximately 1 month).

[0189] Container • Glass Vial: Amber Glass 12 x 32 mm Screw Neck Vial, 2 mL Volume, 100 / pk (Waters, 186000848) • Intravesicle: 250 μL inactivated glass intravesicle (Agilent, 5181-8872) • Cap: Green, 12 x 32 mm Screw Neck Cap and PTFE / Silicone Septum, 100 / pk (Waters, 186002130)

[0190] Subsequently, following the method described in Example 6, cells were stimulated with IL-36α at a final concentration of 50 ng / mL, IL-36β at 10 ng / mL, or IL-36γ at 50 ng / mL, respectively, to assess the inhibitory activity of the antibody. The dilutions for the anti-human IL-36R antibody and the rat isotype control group were obtained by 7 stages of 4-fold dilution, with 100 nM as the maximum treatment concentration. The obtained data were analyzed using GraphPad Prism (registered trademark) Ver8.2.1, and fitted using log(inhibitor) vs. response-variable slope (four parameters). The results are shown in Figure 15.

[0191] The evaluation results showed that, under storage conditions of 37°C for 30 days, the antibody in Example 1 blocked the signaling of IL-36α and IL-36γ, but did not block the signaling of IL-36β, consistent with the results at 4°C. Under the same storage conditions of 37°C for 30 days, no change was observed in the strength or specificity of the antibody's IL-36R signaling blocking activity. Based on these results, the sequence line of this antibody can be considered to have excellent stability. [Industrial Applicability]

[0192] This invention can be used to treat diseases associated with specific information of IL-36R, such as autoimmune diseases or cancer, and its industrial significance is great. [Simplified Explanation of the Diagram]

[0193] [Figure 1] Figure 1 shows the sequence alignment of the amino acid sequences of the active forms of human IL-36α, β, and γ. [Figure 2] Figure 2 shows the amino acid sequence of human IL-36R. [Figure 3] Figure 3 is a schematic diagram showing the three-dimensional structure of human IL-36R. [Figure 4-1] Figures 4-1 and 4-2 show the sequence alignment of the amino acid sequence of human IL-36R containing the signaling peptide with the amino acid sequences of IL-36R from cynomolgus monkeys, rhesus monkeys, mice, and rats. [Figure 4-2] Same as above. [Figure 5A] Figure 5A shows the results of confirming the expression of human IL-36R in cell lines that stably express human IL-36R. [Figure 5B] Figure 5B shows the results of confirming the expression of human IL-1RAcP in 293A cells. [Figure 5C] Figure 5C shows the results of IL-36R performance evaluation in cells confirming human IL-36R function. [Figure 5D] Figure 5D shows the results of IL-1RAcP performance evaluation in cells confirming human IL-36R function. [Figure 6] Figure 6 shows the results of anti-IL-36R antibody binding screening and IL-36α-induced signaling inhibition screening. [Figure 7A] Figure 7A shows the results of IL-36β signaling inhibition relative to IL-36α among the selected anti-IL-36R antibodies. [Figure 7B] Figure 7B shows the results of IL-36γ signaling inhibition relative to IL-36α among the selected anti-IL-36R antibodies. [Figure 8] Figure 8 shows the results of screening for IL-36R neutralizing antibodies that do not inhibit IL-36β signaling. [Figure 9] Figure 9 shows the amino acid sequences of the heavy chain variable region and light chain variable region of the IL-36R message-specific blocking antibody of Experimental Example 1. [Figure 10-1] Figures 10-1 and 10-2 show the dosage response curves of the antibody for human IL-36R variants 1-6 in Reference Examples 1 and 2 and Experimental Example 1. [Figure 10-2] Same as above. [Figure 11] Figures 11(a)-(c) show the evaluation results of the IL-36R message-blocking activity of the antibody purified from the fusion tumor culture supernatant of Experimental Example 1 and Reference Examples 1 and 2. (a) IL-36α, 50 ng / mL, (b) IL-36β, 10 ng / mL, (c) IL-36γ, 50 ng / mL. [Figure 12] Figures 12(a)-(c) show the evaluation results of the IL-36R message-blocking activity of the recombinant antibody of Experimental Example 1 and Reference Examples 1 and 2. (a) IL-36α, 50ng / mL, (b) IL-36β, 50ng / mL, (c) IL-36γ, 50ng / mL.[Figure 13] Figures 13(a) to (c) show the evaluation results of the IL-36R signaling blocking activity of the recombinant antibodies of Test Example 1 and Reference Example 1. (a) IL-36β, 2 ng / mL, (b) IL-36β, 10 ng / mL, (c) IL-36β, 250 ng / mL. [Figure 14] Figures 14(a) to (c) show the evaluation results of the IL-36R signaling blocking activity of the recombinant antibodies of Test Example 1 and Reference Example 1. (a) IL-36α, 50 ng / mL, (b) IL-36β, 10 ng / mL, (c) IL-36γ, 50 ng / mL. [Figure 15] Figures 15(a) to (c) show the evaluation results of the IL-36R signaling blocking activity of the recombinant antibody of Test Example 1 after refrigeration (4°C) and storage at 37°C for 30 days (approximately 1 month). (a) IL-36α, 50ng / mL, (b) IL-36β, 10ng / mL, (c) IL-36γ, 50ng / mL. [Sequence List]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

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[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

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[0280]

[0281]

[0282]

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[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

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[0297]

[0298]

[0299]

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[0302]

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[0310]

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[0321]

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[0325]

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[0330]

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[0332]

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[0340]

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[0345]

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[0350]

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[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

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[0375]

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Claims

1. An antibody against human IL-36R, or a fragment thereof, or a derivative thereof, wherein the antibody is against human IL-36R, or a fragment thereof, or a derivative thereof, wherein... Among the three human IL-36R ligands IL-36α, β and γ, the 50% blocking concentration (IC50) for the message of at least one ligand is more than 10 times that for the message of at least one other ligand.

2. The anti-human IL-36R antibody or fragment thereof, or derivative thereof, as claimed in claim 1, which shows a 50% effective concentration (EC50) of less than 1 × 10⁻⁷ M in a Cell ELISA of cells expressed with IL-36R.

3. The anti-human IL-36R antibody or fragment thereof, or derivative thereof, as claimed in claim 1 or 2, wherein the 50% blocking concentration against the message of the human IL-36R ligand IL-36β is more than 10 times the 50% blocking concentration against the message of the human IL-36R ligands IL-36α and γ.

4. The anti-human IL-36R antibody or fragment thereof, or derivative thereof, as claimed in claim 1 or 2, wherein the signaling of the human IL-36R ligands IL-36α and γ is blocked by more than 50%, and the signaling of IL-36β is blocked by less than 50%.

5. Anti-human IL-36R antibody or fragment thereof, or derivative thereof, as claimed in claim 3 or 4, wherein at a concentration of 0.2 ng / mL or higher, the 50% blocking concentration of IL-36β signaling is 10 × 10⁻⁸ M or higher.

6. An anti-human IL-36R antibody or fragment thereof, or a derivative thereof, as claimed in any of claims 1 to 5, which binds to domain I of human IL-36R.

7. The anti-human IL-36R antibody or fragment thereof, or derivative thereof, as claimed in any of claims 1 to 6, is bound to at least two regions of the amino acid sequence of human IL-36R (Sequence Number 11), namely, the region consisting of amino acid residues 1 to 8 from the N-terminus, the region consisting of amino acid residues 25 to 32, and the region consisting of amino acid residues 81 to 88.

8. The anti-human IL-36R antibody or fragment thereof, or derivative thereof, as claimed in any of claims 1 to 7, is bound to the 4th lysine residue and the 28th isoleucine residue from the N-terminus of the amino acid sequence of human IL-36R (Sequence No. 11).

9. An anti-human IL-36R antibody or fragment thereof, or a derivative thereof, as claimed in any of claims 1 to 8, comprising at least a heavy chain variable sequence, wherein the heavy chain variable sequence (1) comprises an amino acid sequence of sequence number 23 or 25 (XH001 or XH002) as a CDR-H1 sequence, (1b) an amino acid sequence having at least 80% similarity to the amino acid sequence of (1a) above, or (1c) an amino acid sequence in which one or two amino acid residues of the amino acid sequence of (1a) above are substituted, deleted or added; (2) comprises an amino acid sequence of (2a) selected from sequence numbers 27, 29, 31 and 213 (XH003 to XH005 and XH007) as a CDR-H2 sequence, (2b) An amino acid sequence having at least 88% similarity to the amino acid sequence of (2a) above, or (2c) An amino acid sequence in which one or two amino acid residues of the amino acid sequence of (2a) above are substituted, deleted or added; (3) An amino acid sequence comprising (3a) selected from sequence numbers 33, 215 and 217 (XH006, XH008 and XH009) as a CDR-H3 sequence, (3b) an amino acid sequence having at least 83% similarity to the amino acid sequence of (3a) above, or (3c) an amino acid sequence in which one or two amino acid residues of the amino acid sequence of (3a) above are substituted, deleted or added.

10. The anti-human IL-36R antibody or fragment thereof, or derivative thereof, as claimed in claim 9, wherein the frame sequence of the aforementioned heavy chain variable sequence contains frame sequences of various types of immunoglobulins of humans or non-human animals including monkeys, mice or rats.

11. The anti-human IL-36R antibody or fragment thereof, or derivative thereof, as claimed in claim 9 or 10, wherein the aforementioned heavy chain variable sequence comprises (a) an amino acid sequence selected from sequence number 45 (Pro000713v), sequence number 47 (Pro000722v), sequence number 49 (Pro001558v), sequence number 51 (Pro001562v), sequence number 53 (Pro001554v), sequence number 55 (Pro001566v), sequence number 57 (Pro001570v), sequence number 59 (Pro001574v), sequence number 177 (Pro002817v), sequence number 181 (Pro002818v), and sequence number 185 (Pro002819v), (b) an amino acid sequence having at least 80% similarity to the amino acid sequence in (a) above, or (c) an amino acid sequence in which 1 to 12 amino acid residues in the amino acid sequence in (a) above are substituted, deleted or added.

12. The anti-human IL-36R antibody or fragment thereof, or derivative thereof, as claimed in any of claims 1 to 11, comprises at least a light chain variable sequence, wherein the light chain variable sequence comprises (1a) an amino acid sequence selected from sequence numbers 35, 37, and 219 (XL001, XL002, and XL006) as a CDR-L1 sequence, (1b) an amino acid sequence having at least 84% similarity to the amino acid sequence of (1a) above, or (1c) an amino acid sequence in which one or two amino acid residues of the amino acid sequence of (1a) above are substituted, deleted, or added; (2) an amino acid sequence comprising (2a) an amino acid sequence selected from sequence numbers 39, 41, and 221 (XL003, XL004, and XL007) as a CDR-L2 sequence, (2b) An amino acid sequence having at least 85% similarity to the amino acid sequence of (2a) above, or (2c) An amino acid sequence in which one or two amino acid residues of the amino acid sequence of (2a) above are substituted, deleted or added; (3) An amino acid sequence comprising (3a) selected from sequence numbers 43, 223 and 225 (XL005, XL008 and XL009) as a CDR-L3 sequence, (3b) an amino acid sequence having at least 88% similarity to the amino acid sequence of (3a) above, or (3c) an amino acid sequence in which one or two amino acid residues of the amino acid sequence of (3a) above are substituted, deleted or added.

13. The anti-human IL-36R antibody or fragment thereof, or derivative thereof, as claimed in claim 12, wherein the aforementioned light chain variable sequence further contains a framework sequence of various types of immunoglobulins of human or non-human animals including monkeys, mice or rats.

14. The anti-human IL-36R antibody or fragment thereof, or derivative thereof, as claimed in claim 12 or 13, wherein the aforementioned light chain variable sequence comprises (a) an amino acid sequence selected from sequence number 61 (Pro000714v), sequence number 63 (Pro001332v), sequence number 65 (Pro001333v), sequence number 67 (Pro001334v), sequence number 179 (Pro002821v), sequence number 183 (Pro002823v), and sequence number 187 (Pro002822v), (b) an amino acid sequence having at least 80% similarity to the amino acid sequence of (a), or (c) an amino acid sequence in which 1 to 10 amino acid residues of the amino acid sequence of (a) are substituted, deleted, or added.

15. An anti-human IL-36R antibody or fragment thereof, or a derivative thereof, as claimed in any of claims 1 to 14, wherein the heavy chain constant region and / or the light chain constant region are constant regions of various types of immunoglobulins of humans or non-human animals including mice, rats or monkeys.

16. An anti-human IL-36R antibody or fragment thereof, or a derivative thereof, as claimed in any of claims 1 to 15, which is a Fab, scFv, Diabody or bispecific antibody, or a derivative thereof.

17. A nucleic acid molecule comprising a polynucleotide sequence encoding an anti-human IL-36R antibody or a fragment thereof, or a derivative thereof, as claimed in any of claims 1 to 16.

18. A selection vector or expression vector comprising at least one nucleic acid molecule as claimed in claim 17.

19. A recombinant somatic cell having a vector as described in claim 18 introduced therein.

20. A method for producing an anti-human IL-36R antibody or a fragment thereof, or a derivative thereof, as claimed in any one of claims 1 to 16, comprising culturing recombinant cells as claimed in claim 19.

21. A pharmaceutical composition comprising one or more of an anti-human IL-36R antibody or a fragment thereof selected from any one of claims 1 to 16, or derivatives thereof, a nucleic acid molecule as claimed in claim 17, a vector as claimed in claim 18, and a group of recombinant somatic cells as claimed in claim 19.

22. The pharmaceutical composition of claim 21, which is administered to humans to modulate or modify the immune response.

23. The pharmaceutical composition of claim 21 or 22 is administered to humans at a frequency of less than once a week.

24. Any pharmaceutical composition of any of claims 21 to 23 is used for the treatment and / or prevention of cancer or autoimmune diseases in vertebrates.

25. The pharmaceutical composition of any of claims 21 to 24 further contains pharmaceutically permissible diluents, drug carriers, and / or other additives.

26. The pharmaceutical composition of any one of claims 21 to 25 further contains a second active ingredient.

27. The pharmaceutical composition of claim 26, wherein the aforementioned second active ingredient is selected from azelastine, oxatomide, mequitazine, fexofenadine, epinastine, ebastine, cetirizine, levocetirizine, bepotastine, emedastine, olopatadine, loratadine, levocabastine, ozagrel, seratrodast, ramatroban, pranlukast, montelukast, zafirlukast, subplatast, and diphenhydramine. The group consisting of one or more of the following: amine, dimenhydrinate, diphenylpyraline, clemastine, chlorpheniramine, triprolidine, promethazine, alimethazine, hydroxyzine, homochlorcyclizine, cyproheptadine, mesalazine, interferon beta-1b, interferon beta-1a, fingolimod hydrochloride, natalizumab, glatiramer acetate, dimethyl fumarate, etretinate, tacrolimus, mercaptopurine, azathioprine, and apremilast.

28. The pharmaceutical composition of claim 26 or 27, wherein the aforementioned second active ingredient is selected from corticosteroids, antiemetics, ondansetron hydrochloride, granisetron hydrochloride, metroclopramide, domperidone, haloperidol, cyclizine, lorazepam, prochlorperazine, dexamethasone, levomepromazine, tropisetron, cancer vaccines, GM-CSF inhibitors, GM-CSF, DNA vaccines, cell-based vaccines, dendritic cell vaccines, recombinant viral vaccines, heat shock protein (HSP) vaccines, homologous tumor vaccines, autologous tumor vaccines, analgesics, ibuprofen, naproxen. (en), trisalicylic acid choline magnesium, oxycodone hydrochloride, anti-angiogenic drugs, anti-angiogenic drugs, anti-PD-1 antibodies, nivolumab, pembrolizumab, tislelizumab, anti-PD-L1 antibodies, atezolizumab, avelumab, durvalumab, anti-CTLA4 antibodies, ictovir Ipilimumab, anti-CD20 antibody, rituximab, anti-HER2 antibody, trastuzumab, anti-CCR4 antibody, mogamulizumab, anti-VEGF antibody, bevacizumab, anti-VEGF receptor antibody, soluble VEGF receptor fragment, anti-TWEAK antibody, anti-TWEAK receptor antibody, soluble TWEAK receptor fragment, AMG 706, AMG386, Anti-α4β7 antibody, Bevacizumab, Etrolizumab, Anti-SIRPα antibody, Anti-CD40 antibody, Anti-CD40L antibody, Anti-proliferative drugs, Farnesyl protein transferase inhibitors, αvβ3 inhibitors, αvβ5 inhibitors, p53 inhibitors, Kit receptor inhibitors, ret receptor inhibitors, PDGFR inhibitors, Growth hormone secretion inhibitors, Angiopoietin inhibitors, Tumor-infiltrating macrophage inhibitors, c-fms The group includes one or more of the following: inhibitors, anti-c-fms antibodies, CSF-1 inhibitors, anti-CSF-1 antibodies, soluble c-fms fragments, pegvisomant, gemcitabine, panitumumab, irinotecan, TNFα inhibitors, IL-17A inhibitors, IL-17F inhibitors, IL-17RA inhibitors, IL-23p19 inhibitors, IL-23p40 inhibitors, and IL-1RAcP inhibitors, and SN-38.

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