Anti-human interleukin-36 receptor monoclonal antibody and its use

Abstract

The present invention provides an anti-human interleukin-36 receptor (IL-36R) monoclonal antibody and uses thereof. This monoclonal antibody comprises three heavy chain complementarity determining regions (CDR-H1, CDR-H2, and CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2, and CDR-L3). The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are set forth in SEQ ID NOs: 1 to 6, respectively. Compared to spesolimab, this monoclonal antibody has equivalent binding affinity to human IL-36R and superior neutralizing activity at the cellular level.

Description

[Technical Field]

[0001] The present application relates to the field of antibody drugs. Specifically, the present application relates to a monoclonal antibody against human interleukin-36 receptor (IL-36R) and uses thereof. [Background technology]

[0002] Interleukin-36 (IL-36) belongs to the IL-1 family (IL1F) and is composed of the IL-36 receptor agonists IL-36α (IL1F6), IL-36β (IL1F8), and IL-36γ (IL1F9), and the IL-36 receptor antagonist IL-36Ra (IL1F5). Its structural pattern resembles that of the classical IL-1 family: it lacks a signal peptide, cannot be secreted via the classical Golgi-endoplasmic reticulum pathway, and is typically activated by protease hydrolysis after first forming an inactive precursor. IL-36 can be produced by a variety of cells, including monocytes, macrophages, T and B lymphocytes, keratinocytes, and epithelial cells. The IL-36 receptor consists of a specific receptor, IL-36R (also known as IL-1RL2, interleukin-1 receptor-like 2), and an IL-1 receptor accessory protein (IL-1RAcP), each of which consists of three domains: an extracellular domain, a transmembrane domain, and an intracellular Toll / IL-1 receptor (TIR) ​​domain. Proteolytically matured agonist IL-36 ligands (α, β, and γ) bind to IL-36R to form a binary complex, which then recruits IL-1RAcP to form a functional ternary complex. The intracellular TIR of IL-36R and IL-1RAcP interact to aggregate the signaling adapter proteins myeloid differentiation factor 88 (MyD88) and interleukin-1 receptor-associated kinase (IL-1 receptor-associated kinase (IRAK)), activating the downstream NF-κB (Nuclear factor kappa-B) and MAPK (Mitogen-activated protein kinase) signaling pathways. Normal expression of IL-36 can mediate host defense through an inflammatory response.Dysregulated IL-36 expression stimulates receptor-expressing cells (epithelial cells, fibroblasts, keratinocytes, monocytes, macrophages, dendritic cells, and T cells) to produce proinflammatory cytokines, chemokines, and adhesion molecules, which mediate pathological inflammatory responses and are involved in the pathological processes of chronic inflammatory and autoimmune diseases such as generalized pustular psoriasis, palmoplantar pustulosis, atopic dermatitis, inflammatory bowel disease, and chronic obstructive pulmonary disease. [1]Town JE, Garka KE, Renshaw BR, et al.Interleukin (IL)-1F6,IL-1F8,and IL-1F9 signal through IL-1Rrp2 and IL-1RAcP to activate the pathway leading to NF-kappaB and MAPKs [J].Journal of Biological Chemistry,2004,279(14):13677-13688. [2] Gabay C, Towne J E. Regulation and function of interleukin-36 cytokines in homeostasis and pathological conditions [J]. Journal of Leukocyte Biology, 2015, 97(4):645. [3]Henry CM, Sullivan GP, ​​Clancy DM, et al.Neutrophil-Derived Proteases Escalate Inflammation through Activation of IL-36 Family Cytokines[J].Cell Reports,2016,14(4):708-722. [4]Town JE, Renshaw BR, Douangpanya J, et al.Interleukin-36 (IL-36) Ligands Require Processing for Full Agonist (IL-36α,IL-36β,and IL-36γ) or Antagonist (IL-36Ra) Activity [J].Journal of Biological Chemistry,2011,286(49):42594-602. [5]Buhl,AL,Wenzel,J.Interleukin-36 in Infectious and Inflammatory Skin Diseases[J].Frontiers in Immunology,2019.01162 DISCLOSURE OF THE INVENTION

[0003] The present application aims to provide a novel anti-human IL-36R monoclonal antibody, a pharmaceutical composition containing the monoclonal antibody, and pharmaceutical uses of the monoclonal antibody.

[0004] The aspects of the present application are as follows.

[0005] 1. An anti-human IL-36R monoclonal antibody comprising three heavy chain complementarity determining regions (CDR-H1, CDR-H2, and CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2, and CDR-L3), The amino acid sequence of the CDR-H1 (herein, CDR-H1 refers to heavy chain CDR1) is shown in SEQ ID NO: 1 (NYAMG); The amino acid sequence of the CDR-H2 (herein, CDR-H2 refers to heavy chain CDR2) is shown in SEQ ID NO: 2 (YISGGGSAYYASWAKG); The amino acid sequence of the CDR-H3 (herein, CDR-H3 refers to heavy chain CDR3) is shown in SEQ ID NO: 3 (WAIKSYFFGMDL); The amino acid sequence of the CDR-L1 (herein, CDR-L1 refers to light chain CDR1) is shown in SEQ ID NO: 4 (QASEYISSYLA); The amino acid sequence of the CDR-L2 (herein, CDR-L2 refers to light chain CDR2) is shown in SEQ ID NO: 5 (QASTLAS); The monoclonal antibody as described above, wherein the amino acid sequence of the CDR-L3 (herein, CDR-L3 represents light chain CDR3) is set forth in SEQ ID NO: 6 (QTNNAIHTYGGA).

[0006] 2. Contains a heavy chain variable region and a light chain variable region; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7, and the amino acid sequence is EVQLVESGGGLVQPGGSLRLSCAASGIDLSNYAMGWVRQAPGKGLEWVGYISGGGSAYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWAIKSYFFGMDLWGQGTLVTVSS; Item 2. The monoclonal antibody according to Item 1, wherein the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 8, and the amino acid sequence is DIQMTQSPSSVSASVGDRVTITCQASEYISSYLAWYQQKPGKAPKLLIYQASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQTNNAIHTYGGAFGGGTKVEIK.

[0007] 3. An isolated nucleic acid encoding the monoclonal antibody of any one of the preceding claims.

[0008] 4. A host cell comprising the nucleic acid according to item 3.

[0009] The nucleic acid can be present on a vector. The vector can be of any type, for example, a recombinant vector such as an expression vector. Any of several host cells can be used. In one embodiment, the host cell is a prokaryotic cell, for example, E. coli. In another embodiment, the host cell is a eukaryotic cell, for example, a mammalian cell such as a Chinese hamster ovary (CHO) cell.

[0010] 5. A method for producing a monoclonal antibody, comprising culturing the host cell of item 4 to produce the monoclonal antibody of any one of the preceding items.

[0011] The method comprises producing the anti-human IL-36R monoclonal antibody by expressing a recombinant vector encoding the anti-human IL-36R monoclonal antibody in a suitable host cell. In a specific embodiment, the method comprises expressing the anti-human IL-36R monoclonal antibody by culturing a host cell comprising the nucleic acid encoding the anti-human IL-36R monoclonal antibody. The method may further comprise recovering the anti-human IL-36R monoclonal antibody from the host cell culture or host cell culture medium.

[0012] 6. A pharmaceutical composition comprising the monoclonal antibody described in any one of the preceding claims and a pharmaceutically acceptable carrier.

[0013] The pharmaceutical composition may further comprise another therapeutic agent (eg, a different anti-human IL-36R antibody).

[0014] 7. The pharmaceutical composition according to item 6, which is used to treat a disease associated with IL-36R-mediated signaling.

[0015] 8. The disease associated with IL-36R-mediated signaling is selected from dermatitis, psoriasis, inflammatory bowel disease, arthritis, systemic lupus erythematosus, inflammatory lung disease, and chronic kidney disease; Preferably, the disease associated with IL-36R-mediated signaling is selected from generalized pustular psoriasis, palmoplantar pustulosis, atopic dermatitis, inflammatory bowel disease, chronic obstructive pulmonary disease, plaque psoriasis, psoriatic arthritis, multiple sclerosis, rheumatoid arthritis, scleroderma, asthma, and ankylosing spondylitis.

[0016] 9. Use of the monoclonal antibody according to any one of the preceding claims in the preparation of a medicament for treating a disease associated with IL-36R-mediated signaling.

[0017] 10. The disease associated with IL-36R-mediated signaling is selected from dermatitis, psoriasis, inflammatory bowel disease, arthritis, systemic lupus erythematosus, inflammatory lung disease, and chronic kidney disease; Preferably, the disease associated with IL-36R-mediated signaling is selected from generalized pustular psoriasis, palmoplantar pustulosis, atopic dermatitis, inflammatory bowel disease, chronic obstructive pulmonary disease, plaque psoriasis, psoriatic arthritis, multiple sclerosis, rheumatoid arthritis, scleroderma, asthma, and ankylosing spondylitis.

[0018] 11. A method for treating a disease associated with IL-36R-mediated signaling, comprising: The method comprising administering the monoclonal antibody of any one of the preceding claims or the pharmaceutical composition of any one of the preceding claims to a subject in need thereof.

[0019] 12. The disease associated with IL-36R-mediated signaling is selected from dermatitis, psoriasis, inflammatory bowel disease, arthritis, systemic lupus erythematosus, inflammatory lung disease, and chronic kidney disease; Preferably, the disease associated with IL-36R-mediated signaling is selected from generalized pustular psoriasis, palmoplantar pustulosis, atopic dermatitis, inflammatory bowel disease, chronic obstructive pulmonary disease, plaque psoriasis, psoriatic arthritis, multiple sclerosis, rheumatoid arthritis, scleroderma, asthma, and ankylosing spondylitis.

[0020] This application provides a novel anti-human IL-36R monoclonal antibody (Spesolimab) that has the same binding affinity to IL-36R as the conventional anti-human IL-36R monoclonal antibody (Spesolimab) and has superior neutralizing activity at the cellular level. Spesolimab is a monoclonal antibody drug targeting IL-36R, developed by Boehringer Ingelheim. The Phase 3 clinical trial, NAVIGATOR, of Spesolimab for the treatment of adult generalized pustular psoriasis was successful, and the drug has been approved for marketing by the U.S. Food and Drug Administration and has been granted priority review status by the China National Medical Products Administration. The monoclonal antibody of the present application (e.g., QX009N (HZD25-54)) may be an IgG1 (immunoglobulin G1)-type humanized monoclonal antibody targeting IL-36R (interleukin-36 receptor), with high affinity for IL-36R, capable of specifically binding to IL-36R and blocking IL-36 inflammatory pathway signaling. HZD25-54 binds to IL-36R and competitively blocks the binding of receptor agonists (IL-36α, β, γ) to IL-36R, downregulating downstream pro-inflammatory and pro-fibrotic signaling pathways and inhibiting inflammatory responses mediated by epithelial cells, fibroblasts, and immune cells, thereby reducing the release of disease-causing cellular inflammatory factors in inflammatory diseases and skin diseases and achieving the goal of disease control.

[0021] The monoclonal antibody of the present application is expected to exhibit superior neutralizing activity at the cellular level to spesolimab (expressed and prepared according to the sequence disclosed in the patent) and to exhibit good clinical efficacy in the prevention and treatment of related diseases. [Brief explanation of the drawings]

[0022] The accompanying drawings are used for a better understanding of the present application and shall not constitute undue limitations to the present application. [Figure 1]Figure 1 shows the results of nucleic acid electrophoresis for constructing the HZD25-54 transient expression plasmid, where M is a marker; band 1 is the PCR product 25VH-Hu12; band 2 is pQX1, HindIII / BamHI; band 3 is the PCR product 25VK-Hu17; and band 4 is pQX2.3, HindIII / BsiHI. [Figure 2] FIG. 2 is a flow chart of transient expression. [Figure 3] FIG. 3 is an electrophoretic detection diagram of QX009N (HZD25-54). [Figure 4] FIG. 4 shows the activity of QX009N (HZD25-54) and spesolimab analogs in neutralizing human IL-36 (α, β, γ)-induced STAT3 phosphorylation in HT29 reporter gene cells. [Figure 5] FIG. 5 shows the activity of QX009N (HZD25-54) and spesolimab analogs in neutralizing the release of CXCL-1 and IL-8 from HT29 cells induced by human IL-36 (α, β, γ). [Figure 6] FIG. 6 shows the activity of QX009N (HZD25-54) and spesolimab analogs in neutralizing the release of CXCL-1 and IL-8 from A431 cells induced by human IL-36 (α, β, γ). [Figure 7] FIG. 7 shows the activity of QX009N (HZD25-54) and spesolimab analogs in neutralizing human IL-36β-induced IL-8 release from PBMC cells. Summary of the Invention

[0023]

[0033] Exemplary embodiments of the present application will be described below. These embodiments include various details of the embodiments of the present application for ease of understanding, and should be considered as examples only. Therefore, those skilled in the art will recognize that various changes and modifications to the embodiments described herein can be made without departing from the scope and spirit of the present application. In addition, in the following description, for clarity and conciseness, descriptions of well-known functions and structures are omitted.

[0024] Scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art, except that in case of conflict, the definitions used herein shall control.

[0025] Generally speaking, the terms used herein have the following meanings:

[0026] As used herein, an "isolated" antibody refers to an antibody that has been separated from the components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, and the purity is determined, for example, by electrophoresis (e.g., SDS-PAGE isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B848:79-87 (2007).

[0027] As used herein, a "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., each antibody in the population is identical and / or binds to the same epitope. Except for possible variant antibodies (e.g., those containing naturally occurring mutations or those generated during the preparation of a monoclonal antibody), such variants are generally present in minor amounts. Unlike a typical polyclonal antibody preparation, which contains different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody being obtained from a population of substantially homogeneous antibodies and should not be construed as requiring the antibody to be prepared by any particular method. For example, the monoclonal antibodies of the present application can be produced by several techniques, including, but not limited to, hybridoma technology, recombinant DNA technology, phage display technology, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci. This text describes these and other exemplary methods for preparing monoclonal antibodies.

[0028] As used herein, "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, "binding affinity," as used herein, refers to the intrinsic binding affinity that reflects a 1:1 interaction between binding partner members (e.g., an antibody and an antigen). The affinity of molecule X for partner Y can generally be expressed as an equilibrium dissociation constant (KD). Affinity can be measured by routine methods known in the art.

[0029] As used herein, human interleukin-36 receptor (hIL-36R; sometimes abbreviated as IL-36R) refers to a membrane receptor derived from humans, the amino acid sequence of its extracellular domain is shown in SEQ ID NO: 9, and the underlined portion represents the signal peptide. SEQ ID NO:9: MWSLLLCGLSIALPLSVTADGCKDIFMKNEILSASQPFAFNCTFPPITSGEVSVTWYKNSSKIPVSKIIQSRIHQDETWILFLPMEWGDSGVYQCVIKGRDSCHRIHVNLTVFEKHWCDTSIGGLPNLSDEYKQILHLGKDDSLTCHLHFPKSCVLGPIKWYKDCNEIKGERFTVLE TRLLVSNVSAEDRGNYACQAILTHSGKQYEVLNGITVSITERAGYGGSVPKIIYPKNHSIEVQLGTTLIVDCNVTDTKDNTNLRCWRVNNTLVDDYYDESKRIREGVETHVSFREHNLYTVNITFLEVKMEDYGLPFMCHAGVSTAYIILQLPAPDFR

[0030] As used herein, the term "anti-human IL-36R monoclonal antibody" refers to a monoclonal antibody that can bind to human IL-36R with sufficient affinity so that it can be used as a diagnostic and / or therapeutic agent targeting human IL-36R.

[0031] The anti-human IL-36R monoclonal antibody of the present application does not bind to a target unrelated protein. Here, "unrelated protein" refers to a protein other than the target human IL-36R, and "does not bind" here means that, when the binding ability of the anti-human IL-36R monoclonal antibody of the present application to its target human IL-36R is taken as 100%, the binding ability of the anti-human IL-36R monoclonal antibody of the present application to the unrelated protein is less than 10%, for example, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.

[0032] The anti-human IL-36R monoclonal antibody of the present application may not bind to IL-36R of other animal species. Here, "other animal species" refers to animal species other than humans, such as marmosets, cynomolgus monkeys, pigs, dogs, rabbits, rats, mice, and guinea pigs. Here, "does not bind" means that, when the binding ability of the anti-human IL-36R monoclonal antibody of the present application to its target human IL-36R is taken as 100%, the binding ability of the anti-human IL-36R monoclonal antibody of the present application to IL-36R of other animal species is less than 10%, for example, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.

[0033] The human IL-36R monoclonal antibody of the present application has an equilibrium dissociation constant (K D ) can be included.

[0034] The experimental results demonstrate that the anti-human IL-36R monoclonal antibody of the present application can specifically bind to human IL-36R.

[0035] The anti-human IL-36R monoclonal antibody of the present application has many biological activities equivalent to or superior to those of commercially available similar monoclonal antibody products, such as the activity of neutralizing STAT3 phosphorylation in cells induced by human IL-36 (α, β, γ), the activity of neutralizing the release of CXCL-1 and IL-8 from cells induced by human IL-36 (α, β, γ), and the activity of neutralizing the release of IL-8 from human PBMC cells induced by human IL-36β.

[0036] In one specific embodiment, the amino acid sequence of the heavy chain of the anti-human IL-36R monoclonal antibody of the present application may be set forth in SEQ ID NO:10, and the amino acid sequence of the light chain may be set forth in SEQ ID NO:11. SEQ ID NO: 10 EVQLVESGGGLVQPGGSLRLSCAASGIDLSNYAMGWVRQAPGKGLEWVGYISGGGSAYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWAIKSYFFGMDLWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 11 DIQMTQSPSSVSASVGDRVTITCQASEYISSYLAWYQQKPGKAPKLLIYQASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQTNNAIHTYGGAFGGGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Among them, SEQ ID NOs: 10 and 11 are both humanized sequences, and LALA mutations were introduced into the heavy chain constant region to eliminate the ADCC and CDC effects of the antibody.

[0037] As used herein, an "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule that is contained in cells that ordinarily contain the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0038] As used herein, an "isolated nucleic acid encoding an anti-IL-36R monoclonal antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains of the antibody, and includes such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present in one or more locations in a host cell.

[0039] As used herein, "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0040] As used herein, the terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells encompass "transformants" and "transformed cells," and include the primary transformed cell and its progeny (regardless of the number of passages). Progeny may not be entirely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0041] As used herein, "pharmaceutical composition" refers to a product in a form that is effective for the biological activity of the active ingredient contained therein, said composition not containing additional ingredients that are unacceptably toxic to the subject to whom said formulation is administered.

[0042] As used herein, the term "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0043] As used herein, a "monoclonal antibody" is generally a human antibody and can be prepared using techniques well known to those skilled in the art. For example, human antibodies are generally described in van Dijk, MA and van de Winkel, JG, Curr. Opin. Pharmacol. 5:368-374 (2001) and Lonberg, N., Curr. Opin. Immunol. 20:450-459 (2008).

[0044] Antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to stimulate the production of fully human antibodies or intact antibodies with human variable regions in response to antigenic challenge. These animals typically contain some or all of the human immunoglobulin loci, with the endogenous immunoglobulin loci replaced or present extrachromosomally or randomly integrated within the animal. In such transgenic mice, the endogenous immunoglobulin loci are generally inactivated; see Lonberg, N., Nat. Biotech. 23:1117-1125 (2005) for a review of methods for obtaining human antibodies from transgenic animals. See also, e.g., the XENOMOUSE™ technology described in U.S. Patent Nos. 6,075,181 and 6,150,584, the HUMAB® technology described in U.S. Patent No. 5,770,429, the K-MMOUSE® technology described in U.S. Patent No. 7,041,870, and the VELOCIMOUSE® technology described in U.S. Patent Application Publication No. US2007 / 0061900. The human variable regions of intact antibodies generated from such animals can be further modified, for example, by combination with different human constant regions.

[0045] Human antibodies can also be produced by hybridoma-based methods. Human myeloma cells and mouse-human hybrid myeloma cells used to produce human monoclonal antibodies have been described (see, e.g., Kozbor, D., J. Immunol. 133:3001-3005 (1984); Brodeur, B. R. et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York (1987), pp. 51-63; Boerner, P. et al., Immunol. 147:86-95 (1991)). Human antibodies produced by human B cell hybridoma technology are also described in Li, J. et al., Proc. Natl. Acad. Sci. USA 103:3557-3562 (2006). Other methods include those described, for example, in U.S. Patent No. 7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4); 265-268 (which describes human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers, HP and Brandlein, S., Histology and Histopathology 20:927-937 (2005); Vollmers, HP and Brandlein, S., Methods and Findings in Experimental and Clinical Pharmacology 27:185-191 (2005).

[0046] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries, and then combining such variable domain sequences with desired human constant domains.

[0047] Human antibodies can also be selected based on autoantibody libraries.That is, human antibodies can be isolated by screening for antibodies with one or more desired activities in combinatorial libraries.For example, various methods are known in the art for creating phage display libraries and screening such libraries for antibodies with desired binding properties. This method is reviewed, for example, in Hoogenboom, H.R. et al., Methods in Molecular Biology 178:1-37 (2001), and further described, for example, in McCafferty, J. et al., Nature 348:552-554 (1990); Clackson, T. et al., Nature 352:624-628 (1991); Marks, J.D. et al., J.Mol.Biol. 222:581-597 (1992); Marks, J.D. and Bradbury, A., Methods in Molecular Biology 248:161-175 (2003); Sidhu, S.S. et al., J.Mol.Biol. 338:299-310 (2004); Lee, C.V. et al. al., J. Mol. Biol. 340:1073-1093 (2004); Fellouse, FA, Proc. Natl. Acad. Sci. USA 101:12467-12472 (2004); and Lee, CV et al., J. Immunol. Methods 284:119-132 (2004).

[0048] In some phage display methods, complete sets of VH and VL genes are cloned by polymerase chain reaction (PCR) and randomly recombined into a phage library, which is then screened for antigen-binding phages, as described in Winter, G. et al., Ann. Rev. Immunol. 12:433-455 (1994). Phages typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high-affinity antibodies to immunogens without the need for hybridoma construction. Alternatively, as described in Griffiths, AD et al., EMBO J, 12:725-734 (1993), unimmunized repertoires (e.g., from humans) can be cloned to provide a single source of antibodies against multiple non-self and self antigens in the absence of immunization. Finally, non-immunized libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells, encoding highly variable CDR3 regions using PCR primers containing random sequences, and rearranging them in vitro, as described by Hoogenboom, H.R. and Winter, G., J. Mol. Biol. 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373 and U.S. Patent Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0049] The antibody may also be a multispecific antibody, such as a bispecific antibody. Bispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. Techniques for producing multispecific antibodies include, but are not limited to, recombinant coexpression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein, C. and Cuello, A.C., Nature 305:537-540 (1983); WO93 / 08829; and Traunecker, A. et al., EMBO J. 10:3655-3659 (1991)) and "protuberance-into-cavity" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be produced by various techniques, including engineered electrostatic steering effects to generate antibody Fc heterodimeric molecules (WO 2009 / 089004), cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980 and Brennan, M. et al., Science 229:81-83 (1985)), using leucine zippers to generate bispecific antibodies (see, e.g., Kostelny, S.A. et al., J. Immunol. 148:1547-1553 (1992)), using "double antibody" technology to generate bispecific antibody fragments (see, e.g., Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)), and the use of single-chain Fv (scFv) dimers (see, e.g., Gruber, M. et al. al., J. Immunol. 152:5368-5374 (1994)), and preparation of trispecific antibodies (see, for example, Tutt, A. et al., J. Immunol. 147:60-69 (1991)).

[0050] The monoclonal antibodies described herein also include engineered modified antibodies with three or more functional antigen binding sites, including "octopus antibodies" (see, e.g., US 2006 / 0025576).

[0051] The antibodies herein can also include multispecific antibodies described in WO2009 / 080251, WO2009 / 080252, WO2009 / 080253, WO2009 / 080254, WO2010 / 112193, WO2010 / 115589, WO2010 / 136172, WO2010 / 145792, and WO2010 / 145793, WO2011 / 117330, WO2012 / 025525, WO2012 / 025530, WO2013 / 026835, WO2013 / 026831, WO2013 / 164325, or WO2013 / 174873.

[0052] The monoclonal antibodies described herein may be antibody variants, for example, if it is desired to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletion, insertion, and / or substitution of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, as long as the final construct possesses the desired properties, such as antigen binding. Thus, in certain embodiments, antibody variants with one or more amino acid substitutions are provided. Target sites for substitution mutations include HVRs and FRs. For example, amino acid substitutions can be introduced into a target antibody and products with the desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC, can be screened. [Example]

[0053] All experimental methods used in the following examples are conventional unless otherwise specified.

[0054] Unless otherwise specified, materials, reagents, etc. used in the following examples are obtained from commercial sources.

[0055] Example 1 Preparation of anti-human IL-36R monoclonal antibody QX009N New Zealand rabbits were immunized with Qyuns's self-produced and expressed human interleukin-36 receptor (hIL-36R-Rabbit Fc) as an immunogen, and B cell cloning techniques were used to obtain specific antibody clones. Monoclonal antibodies binding to human IL-36R and exhibiting human IL-36R inhibitory activity were then screened. Cell supernatants were analyzed and screened using binding ELISA and IL-8 release assays from HT29 cells, and the desired clones were selected. The above immunization and screening processes were completed by a commercial company.

[0056] Ten clones were selected for recombinant expression and sequenced. The assay revealed that clone 25# had the best cell-neutralizing activity. Therefore, clone 25# was humanized. NCBI IgBlast was used to perform a homology comparison of human IgG germline sequences. IgGHV3-66*01 was selected as the heavy chain CDR-grafting template, and the CDR regions of clone 25#'s heavy chain (i.e., CDR-H1 (SEQ ID NO: 1), CDR-H2 (SEQ ID NO: 2), and CDR-H3 (SEQ ID NO: 3)) were grafted into the framework region of IgGHV3-66*01. IGKV1-12*01 was selected as the light chain CDR-grafting template, and the CDR regions of clone 25#'s light chain (i.e., CDR-L1 (SEQ ID NO: 4), CDR-L2 (SEQ ID NO: 5), and CDR-L3 (SEQ ID NO: 6)) were grafted into the framework region of IGKV1-12*01. Back mutations were performed at specific sites in the framework regions, and site-directed mutations were performed in the CDR regions to obtain the variable regions of the monoclonal antibody QX009N of the present application. Finally, the sequence of the humanized heavy chain variable region is shown in SEQ ID NO:7, and the amino acid sequence of the humanized light chain variable region is shown in SEQ ID NO:8.

[0057] The heavy chain (SEQ ID NO: 10) gene and light chain variable region (SEQ ID NO: 8) gene were obtained by PCR amplification. The heavy chain expression plasmid pQX1 was double-digested with HindIII and BamHI, and the light chain expression plasmid pQX2.3 was double-digested with HindIII and BsiWI. The PCR-amplified genes were then inserted into the corresponding expression plasmids using infusion recombinase to construct the heavy chain expression plasmid pQX2.1-25VH-Hu12 and the light chain expression plasmid pQX2.3-25VK-Hu17.

[0058] The results of detecting the PCR-amplified heavy chain gene fragment, light chain variable region gene fragment, and double-digested plasmid by nucleic acid electrophoresis are shown in Figure 1. As can be seen from the results in Figure 1, the results of PCR amplification of the antibody heavy and light chain variable regions and double-digestion of the heavy chain expression plasmid and light chain expression plasmid show that the sizes of the heavy chain and light chain plasmids are approximately 5000 bp, the heavy chain is approximately 1469 bp, and the light chain variable region is approximately 441 bp.

[0059] ExpiCHO-S cells were co-transfected with the sequence-correct heavy chain expression plasmid pQX2.1-25VH-Hu12 (the amino acid sequence of the heavy chain expressed thereby is shown in SEQ ID NO: 10) and the sequence-correct light chain expression plasmid pQX2.3-25VK-Hu17 (the amino acid sequence of the light chain expressed thereby is shown in SEQ ID NO: 11). The day before transfection, 3 x 10 ExpiCHO-S cells were cultured for pre-transfection passage. 6 On the day of transfection, the cell density was adjusted to 6 × 10 6 The cells were diluted to 125 ml / ml and 25 ml of cells were placed in a 125 ml shake flask for transfection. The transfection and expression process is shown in Figure 2.

[0060] Six days after transfection, the culture supernatant was collected and purified in one step using Protein A. The purified antibody was detected by SDS-PAGE electrophoresis and designated QX009N (HZD25-54). The results of protein electrophoresis of this antibody are shown in Figure 3. Protein electrophoresis was performed on a denaturing, reducing gel. As can be seen from the results shown in Figure 3, two bands were observed, with sizes of approximately 50 kDa and 25 kDa, respectively, consistent with the theoretical molecular weights of the heavy chain (49.0 kDa) and light chain (23.4 kDa).

[0061] Example 2 Equilibrium dissociation constant (K D ) measurement The affinity of QX009N (HZD25-54) to human IL-36R was detected using a Biacore T200, and all processes were performed at 25°C. A commercially available Protein A chip was used, and an appropriate amount of antibody was immobilized by the capture method to achieve an Rmax of approximately 50 RU and a capture flow rate of 10 μl / min. The antigen was serially diluted, and the instrument flow rate was switched to 30 μl / min. The reference channel and antibody-immobilized channel were run in order of low to high concentration, with buffer as a negative control. After each binding and dissociation, the chip was regenerated with pH 1.5 glycine. Using the instrument's software, a 1:1 binding model was selected and fitted in the Kinetics option to determine the antibody binding rate constant, k. a , dissociation rate constant k d , and the equilibrium dissociation constant K D The value of was calculated.

[0062] We also compared the affinity of QX009N (HZD25-54) with that of spesolimab, a monoclonal antibody against human IL-36R currently undergoing phase 3 clinical trials. The detection method for the known antibody was the same as that for QX009N. The results are shown in Table 1. Here, spesolimab was produced in-house by transiently transfecting ExpiCHO-S cells with an expression plasmid constructed based on the B6 sequence provided in patent US9023995B2. [Table 1] The data in the table are the average values ​​calculated from three tests of each sample.

[0063] Example 3 Activity of QX009N (HZD25-54) and Spesolimab Analogues in Neutralizing Human IL-36 (α, β, γ)-Induced STAT3 Phosphorylation in HT29 Reporter Gene Cells The activity of QX009N (HZD25-54) in antagonizing human IL-36 (α, β, γ)-mediated STAT3 phosphorylation via IL-36R-IL-1RAcp was measured using the HT29 reporter gene cell line as follows: 40,000 cells were added to each well of a 96-well cell culture plate in a volume of 100 μl and cultured overnight at 37°C, 5% CO2. Antibodies were added to the cells and incubated for 1 hour. The final antibody concentrations were 0–10,000 ng / ml. After incubation, 50 μl of a recombinant human IL-36 mixture (containing 2 ng / ml recombinant human IL-36α, 1 ng / ml recombinant human IL-36β, and 40 ng / ml recombinant human IL-36γ) was added per well and cultured for 24 hours at 37°C, 5% CO2. The cell culture supernatant was discarded, and 120 μl of ONE Glo Luciferase Reagent was added to each well and allowed to react for 10 minutes. 80 μl of the solution was transferred to a white 96-well plate, and the luminescence signal was measured. A dose-response curve was plotted to analyze the antagonistic activity of the antibody. The dose-response curve is shown in Figure 4.

[0064] As can be seen from the results shown in Figure 4, QX009N (HZD25-54) can inhibit the phosphorylation of STAT3 in HT29 reporter gene cells induced by human IL-36 (α, β, γ), and the IC of the activity of QX009N (HZD25-54) to inhibit the phosphorylation of STAT3 in HT29 reporter gene cells induced by human IL-36 (α, β, γ) was 50The IC50 of spesolimab analogs was 2.16 ng / ml, indicating their activity in inhibiting STAT3 phosphorylation in HT29 reporter gene cells induced by human IL-36 (α, β, γ). 50 is 15.67ng / ml.

[0065] Example 4: Activity of QX009N (HZD25-54) and spesolimab analogs in neutralizing human IL-36 (α, β, γ)-induced release of CXCL-1 and IL-8 from HT29 cells The activity of QX009N (HZD25-54), which antagonizes human IL-36 (α, β, γ) and induces CXCL-1 and IL-8 release from HT29 cells via IL-36R-IL-1RAcp, was measured using the HT29 cell line as follows: 40,000 cells were added to each well of a 96-well cell culture plate in a volume of 100 μl and cultured overnight at 37°C, 5% CO2. Antibodies were added to the cells and incubated for 1 hour. The final antibody concentrations were 0–10,000 ng / ml. After incubation, 50 μl of a recombinant human IL-36 mixture (containing 1 ng / ml recombinant human IL-36α, 0.2 ng / ml recombinant human IL-36β, and 4 ng / ml recombinant human IL-36γ) was added per well and cultured for 24 hours at 37°C, 5% CO2. The cell culture supernatants were collected, and the expression of CXCL-1 and IL-8 in the supernatants was detected using a sandwich ELISA. The dose-response curves were plotted to analyze the antagonistic activity of the antibodies. The dose-response curves are shown in Figure 5.

[0066] As can be seen from the results shown in Figure 5, QX009N (HZD25-54) can inhibit the release of CXCL-1 and IL-8 from HT29 cells induced by human IL-36 (α, β, γ), and the IC 50 The IC values ​​of spesolimab analogs were 2.21 ng / ml and 1.53 ng / ml, respectively, and they inhibited the release of CXCL-1 and IL-8 from HT29 cells induced by human IL-36 (α, β, γ). 50are 12.29 ng / ml and 11.47 ng / ml, respectively.

[0067] Example 5: Activity of QX009N (HZD25-54) and spesolimab analogues in neutralizing human IL-36 (α, β, γ)-induced release of CXCL-1 and IL-8 from A431 cells The activity of QX009N (HZD25-54), which antagonizes human IL-36 (α, β, γ) and induces the release of CXCL-1 and IL-8 from A431 cells via IL-36R-IL-1RAcp, was measured using the A431 cell line as follows: 40,000 cells were added to each well of a 96-well cell culture plate in a volume of 100 μl and cultured overnight at 37°C, 5% CO2. Antibodies were added to the cells and incubated for 1 hour, with final antibody concentrations ranging from 0 to 10,000 ng / ml. After incubation, 50 μl of a recombinant human IL-36 mixture (containing 20 ng / ml recombinant human IL-36α, 2 ng / ml recombinant human IL-36β, and 50 ng / ml recombinant human IL-36γ) was added per well and cultured for 24 hours at 37°C, 5% CO2. The cell culture supernatants were collected, and the expression of CXCL-1 and IL-8 in the supernatants was detected using a sandwich ELISA. The antagonistic activity of the antibodies was analyzed by plotting a dose-response curve, which is shown in Figure 6.

[0068] As can be seen from the results shown in Figure 6, QX009N (HZD25-54) can inhibit the release of CXCL-1 and IL-8 from A431 cells induced by human IL-36 (α, β, γ), and the IC 50 The IC values ​​of spesolimab analogs were 4.12 ng / ml and 2.89 ng / ml, respectively, and these values ​​were significant for inhibiting the release of CXCL-1 and IL-8 from A431 cells induced by human IL-36 (α, β, γ). 50 are 22.46ng / ml and 16.02ng / ml, respectively.

[0069] Example 6: Activity of QX009N (HZD25-54) and Spesolimab Analogues in Neutralizing Human IL-36β-Induced IL-8 Release from Human PBMC Cells The activity of QX009N (HZD25-54) to antagonize human IL-36β, which induces IL-8 release from human PBMC cells via IL-36R-IL-1RAcp, was measured using human PBMC cells as follows. PBMCs were isolated from human venous blood (collected from healthy adult volunteers) by density gradient centrifugation. 20,000 cells were added to each well of a 96-well cell culture plate in a volume of 100 μl. Antibody was then added to the PBMCs and incubated for 1 h. The final antibody concentrations ranged from 0 to 5,000 ng / ml. After incubation, 50 μl / well of 10 ng / ml recombinant human IL-36β was added and the cells were cultured at 37°C and 5% CO2 for 24 h. Cell culture supernatants were collected, and IL-8 expression in the supernatants was detected using a sandwich ELISA. A dose-response curve was plotted to analyze the antagonistic activity of the antibody. The dose-response curve is shown in Figure 7.

[0070] As can be seen from the results shown in Figure 7, QX009N (HZD25-54) can inhibit human IL-36β-induced IL-8 release from human PBMC cells, and the IC 50 The IC50 of the spesolimab analogue was 4.69 ng / ml, which is the IC50 of the activity of the spesolimab analogue in inhibiting human IL-36β-induced IL-8 release from human PBMC cells. 50 is 26.59ng / ml.

Claims

1. An anti-human interleukin-36 receptor (IL-36R) monoclonal antibody comprising three heavy chain complementarity determining regions, CDR-H1, CDR-H2, and CDR-H3, and three light chain complementarity determining regions, CDR-L1, CDR-L2, and CDR-L3, The amino acid sequence of the CDR-H1 is shown in SEQ ID NO: 1; The amino acid sequence of the CDR-H2 is shown in SEQ ID NO:2; The amino acid sequence of the CDR-H3 is shown in SEQ ID NO:3; The amino acid sequence of the CDR-L1 is shown in SEQ ID NO:4; The amino acid sequence of the CDR-L2 is shown in SEQ ID NO:5; The monoclonal antibody, wherein the amino acid sequence of the CDR-L3 is set forth in SEQ ID NO:

6.

2. comprising a heavy chain variable region and a light chain variable region, The amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:7; The monoclonal antibody of claim 1 , wherein the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:

8.

3. An isolated nucleic acid encoding the monoclonal antibody of claim 1 or 2.

4. A host cell comprising the nucleic acid of claim 3.

5. A method for producing a monoclonal antibody, comprising culturing the host cell of claim 4 to produce the monoclonal antibody of claim 1 or 2.

6. A pharmaceutical composition comprising the monoclonal antibody of claim 1 or 2 and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, which is used to treat a disease associated with IL-36R-mediated signal transduction.

8. the disease associated with IL-36R-mediated signaling is selected from dermatitis, psoriasis, inflammatory bowel disease, arthritis, systemic lupus erythematosus, inflammatory lung disease, and chronic kidney disease; The pharmaceutical composition according to claim 7, wherein the disease associated with IL-36R-mediated signaling is preferably selected from generalized pustular psoriasis, palmoplantar pustulosis, atopic dermatitis, inflammatory bowel disease, chronic obstructive pulmonary disease, plaque psoriasis, psoriatic arthritis, multiple sclerosis, rheumatoid arthritis, scleroderma, asthma, or ankylosing spondylitis.

9. Use of the monoclonal antibody of claim 1 or 2 in the preparation of a medicament for treating a disease associated with IL-36R-mediated signaling.

10. the disease associated with IL-36R-mediated signaling is selected from dermatitis, psoriasis, inflammatory bowel disease, arthritis, systemic lupus erythematosus, inflammatory lung disease, and chronic kidney disease; The use according to claim 9, wherein the disease associated with IL-36R-mediated signaling is selected from generalized pustular psoriasis, palmoplantar pustulosis, atopic dermatitis, inflammatory bowel disease, chronic obstructive pulmonary disease, plaque psoriasis, psoriatic arthritis, multiple sclerosis, rheumatoid arthritis, scleroderma, asthma, and ankylosing spondylitis.

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