Anti-human interleukin 36 receptor monoclonal antibodies and uses thereof

Abstract

The application provides an anti-human interleukin 36 receptor (IL-36R) monoclonal antibody and an application thereof. The anti-human interleukin 36 receptor (IL-36R) monoclonal antibody provided by the application 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), wherein (a) the amino acid sequence of CDR-H1 is shown as SEQ ID NO:1; (b) the amino acid sequence of CDR-H2 is shown as SEQ ID NO:2; (c) the amino acid sequence of CDR-H3 is shown as SEQ ID NO:3; (d) the amino acid sequence of CDR-L1 is shown as SEQ ID NO:4; (e) the amino acid sequence of CDR-L2 is shown as SEQ ID NO:5; and (f) the amino acid sequence of CDR-L3 is shown as SEQ ID NO:6. The anti-human interleukin 36 receptor (IL-36R) monoclonal antibody has the same affinity to human IL-36R as Spesolimab (prepared according to the patent publication sequence table), and has better neutralization activity at the cell level than Spesolimab, and is expected to exhibit good clinical effects in the prevention and treatment of related diseases.

Description

Technical Field

[0001] This application relates to the field of antibody drugs. Specifically, this application relates to a monoclonal antibody targeting the human interleukin 36 receptor (IL-36R) and its application. Background Technology

[0002] Interleukin-36 (IL-36) belongs to the IL-1 family (IL-1F) and is composed of the IL-36 receptor agonists IL-36α (IL-1F6), IL-36β (IL-1F8), and IL-36γ (IL-1F9) and the IL-36 receptor antagonist IL-36Ra (IL-36receptor antagonist, IL-1F5). Its structural pattern is similar to that of the classic IL-1 family: it lacks a signal peptide and cannot be secreted via the classic Golgi-endoplasmic reticulum pathway. It typically forms an inactive precursor first, which is then activated by protease hydrolysis. IL-36 can be produced by various cell types, including monocytes, macrophages, T / B lymphocytes, keratinocytes, and epithelial cells. The receptor for IL-36 consists of the specific receptor IL-36R (also known as IL-1RL2, Interleukin-1 receptor-like2) and the IL-1 receptor accessory protein IL-1RAcP (IL-1 receptor accessory protein). Both are composed of an extracellular domain consisting of three domains: a transmembrane domain and an intracellular Toll / IL-1 receptor (TIR) ​​domain. The proteolytically matured agonistic IL-36 ligands (α, β, γ) bind to IL-36R to form a binary complex, which in turn recruits IL-1RAcP to assemble into a functional ternary complex. The intracellular TIR interaction between IL-36R and IL-1RAcP aggregates the transduction adaptor protein myeloid differentiation factor 88 (MyD88) and interleukin-1 receptor associated kinase (IRAK), thereby activating the downstream NF-κB (Nuclear factor kappa-B) and MAPK (Mitogen-activated protein kinase) signaling pathways. When IL-36 is expressed normally, it can serve as a host defense mechanism through inflammatory responses. When IL-36 expression is dysregulated, it stimulates its receptor-expressing cells (epithelial cells, fibroblasts, keratinocytes, monocytes, macrophages, dendritic cells, and T cells) to produce inflammatory cytokines, chemokines, and adhesion molecules, thereby mediating pathological inflammatory responses and participating 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.

[0003] [1]Towne J E,Garka K E,Renshaw B R,et al.Interleukin(IL)-1F6,IL-1F8,and IL-1F9 signal through IL-1Rrp2 and IL-1RAcP to activate thepathwayleading to NF-kappaB and MAPKs[J].Journal of Biological Chemistry,2004,279(14):13677-13688.

[0004] [2]Gabay C,Towne J E.Regulation and function of interleukin-36cytokines in homeostasis and pathological conditions[J].Journal ofLeukocyteBiology,2015,97(4):645.

[0005] [3]Henry C M,Sullivan G P,Clancy D M,et al.Neutrophil-DerivedProteases Escalate Inflammation through Activation of IL-36FamilyCytokines[J].Cell Reports,2016,14(4):708-722.

[0006] [4]Towne J E,Renshaw B R,Douangpanya J,et al.Interleukin-36(IL-36)Ligands Require Processing for Full Agonist(IL-36α,IL-36β,and IL-36γ)orAntagonist(IL-36Ra)Activity[J].Journal of Biological Chemistry,2011,286(49):42594-602.

[0007] [5]Buhl,AL,Wenzel,J.Interleukin-36in Infectious and InflammatorySkinDiseases[J].Frontiers in Immunology,2019.01162 Summary of the Invention

[0008] The purpose of this application is to provide a novel anti-human IL-36R monoclonal antibody, a pharmaceutical composition comprising the monoclonal antibody, and the pharmaceutical use of the monoclonal antibody.

[0009] The technical solution of this application is as follows:

[0010] 1. A monoclonal antibody against human IL-36R, 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), wherein:

[0011] The amino acid sequence of CDR-H1 (in this specification, CDR-H1 represents heavy chain CDR1) is shown in SEQ ID NO: 1 (NYAMG);

[0012] The amino acid sequence of CDR-H2 (in this specification, CDR-H2 represents heavy chain CDR2) is shown in SEQ ID NO: 2 (YISGGGSAYYASWAKG);

[0013] The amino acid sequence of CDR-H3 (in this specification, CDR-H3 represents heavy chain CDR3) is shown in SEQ ID NO: 3 (WAIKSYFFGMDL);

[0014] The amino acid sequence of CDR-L1 (in this specification, CDR-L1 represents light chain CDR1) is shown in SEQ ID NO: 4 (QASEYISSYLA);

[0015] The amino acid sequence of CDR-L2 (in this specification, CDR-L2 represents light chain CDR2) is shown in SEQ ID NO: 5 (QASTLAS);

[0016] The amino acid sequence of CDR-L3 (in this specification, CDR-L3 represents light chain CDR3) is shown in SEQ ID NO: 6 (QTNNAIHTYGGA).

[0017] 2. The monoclonal antibody according to claim 1, comprising a heavy chain variable region and a light chain variable region, wherein,

[0018] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7. Its amino acid sequence is EVQLVESGGGLVQPGGSLRLSCAASGIDLSNYAMGWVRQAPGKGLEWVGYISGGGSAYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWAIKSYFFGMDLWGQGTLVTVSS;

[0019] The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8, and its amino acid sequence is DIQMTQSPSSVSASVGDRVTITCQASEYISSYLAWYQQKPGKAPKLLIYQASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQTNNAIHTYGGAFGGGTKVEIK.

[0020] 3. An isolated nucleic acid encoding any of the aforementioned monoclonal antibodies.

[0021] 4. A host cell comprising the nucleic acid as described in item 3.

[0022] 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 a variety of host cells can be used. In one embodiment, the host cell is a prokaryotic cell, for example, *Escherichia coli* (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.

[0023] 5. A method for producing a monoclonal antibody, the method comprising culturing a host cell according to claim 4 to produce any of the aforementioned monoclonal antibodies.

[0024] The method includes producing the monoclonal antibody by expressing a recombinant vector encoding the anti-human IL-36R monoclonal antibody in suitable host cells. In some embodiments, the method includes culturing host cells containing nucleic acids encoding the anti-human IL-36R monoclonal antibody to express the nucleic acids. The method may further include recovering the anti-human IL-36R monoclonal antibody from a host cell culture or host cell culture medium.

[0025] 6. A pharmaceutical composition comprising any of the aforementioned monoclonal antibodies and a pharmaceutically acceptable carrier.

[0026] The pharmaceutical composition may further comprise additional therapeutic agents (e.g., different anti-human IL-36R antibodies).

[0027] 7. The pharmaceutical composition according to item 6, for treating IL-36R-mediated signal transduction-related diseases.

[0028] 8. The pharmaceutical composition according to claim 7, wherein the IL-36R-mediated signal transduction-related diseases are selected from dermatitis, psoriasis, inflammatory bowel disease, arthritis, systemic lupus erythematosus, inflammatory lung disease, and chronic kidney disease.

[0029] Preferably, the IL-36R-mediated signal transduction-related diseases are selected from generalized pustular psoriasis, palmoplantar pustulosis, atopic dermatitis, inflammatory bowel disease, chronic obstructive pulmonary disease, psoriasis vulgaris, psoriatic arthritis, multiple sclerosis, rheumatoid arthritis, scleroderma, asthma, or ankylosing spondylitis.

[0030] 9. Use of any of the aforementioned monoclonal antibodies in the preparation of drugs for the treatment of IL-36R-mediated signal transduction-related diseases.

[0031] 10. The use according to item 9, wherein the diseases associated with the IL-36R-mediated signal transduction are selected from dermatitis, psoriasis, inflammatory bowel disease, arthritis, systemic lupus erythematosus, inflammatory lung disease, and chronic kidney disease.

[0032] Preferably, the IL-36R-mediated signal transduction-related diseases are selected from generalized pustular psoriasis, palmoplantar pustulosis, atopic dermatitis, inflammatory bowel disease, chronic obstructive pulmonary disease, psoriasis vulgaris, psoriatic arthritis, multiple sclerosis, rheumatoid arthritis, scleroderma, asthma, or ankylosing spondylitis.

[0033] 11. A method for treating diseases related to IL-36R-mediated signal transduction, comprising:

[0034] Administer to the subject who requires the use of the monoclonal antibody or the pharmaceutical composition described in any of the preceding claims.

[0035] 12. The method according to claim 11, wherein the IL-36R-mediated signal transduction-related diseases are selected from dermatitis, psoriasis, inflammatory bowel disease, arthritis, systemic lupus erythematosus, inflammatory lung disease, and chronic kidney disease.

[0036] Preferably, the IL-36R-mediated signal transduction-related diseases are selected from generalized pustular psoriasis, palmoplantar pustulosis, atopic dermatitis, inflammatory bowel disease, chronic obstructive pulmonary disease, psoriasis vulgaris, psoriatic arthritis, multiple sclerosis, rheumatoid arthritis, scleroderma, asthma, or ankylosing spondylitis.

[0037] This application provides a novel anti-human IL-36R monoclonal antibody that exhibits comparable affinity for IL-36R to the existing anti-human IL-36R monoclonal antibody, Spesolimab, and superior neutralizing activity at the cellular level. It should be noted that Spesolimab is a monoclonal antibody drug targeting IL-36R developed by Boehringer Ingelheim. Spesolimab achieved success in the Phase III NAVIGATOR clinical trial for the treatment of adult generalized pustular psoriasis and has received marketing approval from the U.S. Food and Drug Administration (FDA) and priority review status from the China National Medical Products Administration (NMPA). The monoclonal antibody of this application (e.g., QX009N(HZD25-54)) can be a humanized monoclonal antibody targeting IL-36R (interleukin-36 receptor) of the IgG1 (immunoglobulin G1) type. It has high affinity for IL-36R, specifically binds to IL-36R, and blocks IL-36 inflammatory pathway signaling. HZD25-54 binds to IL-36R, competitively blocking the binding of receptor agonists (IL-36α, β, γ) to IL-36R, downregulating downstream pro-inflammatory and pro-fibrotic signaling pathways, and inhibiting epithelial / fibroblast / immune cell-mediated inflammatory responses. This reduces the release of pathogenic inflammatory factors in inflammatory / skin diseases, thereby achieving the goal of controlling the disease.

[0038] The monoclonal antibody of this application exhibits superior neutralizing activity at the cellular level compared to Spesolimab (prepared by expression according to the disclosed sequence in the patent), and is expected to demonstrate good clinical efficacy in the prevention and treatment of related diseases. Attached Figure Description

[0039] The accompanying drawings are provided to better understand this application and do not constitute an undue limitation thereof. Wherein:

[0040] Figure 1 This is a graph showing the nucleic acid electrophoresis results of the constructed HZD25-54 transient transgenic expression plasmid. In the graph, M: Marker; Band 1: PCR product 25VH-Hu12; Band 2: pQX1, HindIII / BamHI; Band 3: PCR product 25VK-Hu17; Band 4: pQX2.3, HindIII / BsiWI.

[0041] Figure 2 It is a flowchart for instantaneous transition.

[0042] Figure 3 This is the electrophoresis detection image of QX009N(HZD25-54).

[0043] Figure 4This is a graph showing the STAT3 phosphorylation activity induced by QX009N (HZD25-54) and Spesolimab analogs in HT29 reporter cells induced by human IL-36 (α, β, γ).

[0044] Figure 5 This is a graph showing the release of CXCL-1 and IL-8 from HT29 cells induced by QX009N (HZD25-54) and Spesolimab analogues and human IL-36 (α, β, γ).

[0045] Figure 6 This is a graph showing the release of CXCL-1 and IL-8 from A431 cells induced by QX009N (HZD25-54) and Spesolimab analogues and human IL-36 (α, β, γ).

[0046] Figure 7 This is a graph showing the neutralization of IL-8 release activity induced by human IL-36β in PBMC cells by QX009N (HZD25-54) and Spesolimab analogues. Detailed Implementation

[0047] The following description provides exemplary embodiments of this application, including various details to aid understanding, and should be considered merely exemplary. Therefore, those skilled in the art will recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of this application. Similarly, for clarity and brevity, descriptions of well-known functions and structures are omitted in the following description.

[0048] The technical terms mentioned in this specification have the same meanings as those commonly understood by those skilled in the art, and in case of any conflict, the definitions in this specification shall prevail.

[0049] Generally speaking, the terms used in this specification have the following meanings.

[0050] In this specification, an “isolated” antibody is an antibody that has been separated from components of its native environment. In some embodiments, the antibody is purified to a purity greater than 95% or 99%, which is determined by, for example, electrophoresis (e.g., SDS-PAGE isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for evaluating antibody purity, see, for example, Flatman et al., J. Chromatogr. B848: 79-87 (2007).

[0051] In this specification, "monoclonal antibody" means an antibody derived from a substantially homologous group of antibodies, i.e., the individual antibodies constituting the group are identical and / or bind to the same epitopes, and such variants are typically present in trace amounts, except for possible variant antibodies (e.g., containing naturally occurring mutations or generated during the production of monoclonal antibody articles). Unlike polyclonal antibody articles, which typically comprise different antibodies targeting different determinants (epitaxes), each monoclonal antibody in a monoclonal antibody article targets a single determinant on an antigen. Therefore, the modifier "monoclonal" indicates that the antibody is derived from a substantially homologous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, the monoclonal antibody used according to this application can be prepared by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods using transgenic animals containing all or part of human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0052] In this specification, "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 reflecting a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can generally be determined by the equilibrium dissociation constant (K0). D Affinity can be measured using common methods known in the art.

[0053] In this specification, the human interleukin-36 receptor (hIL-36R, sometimes abbreviated as IL-36R) refers to a human membrane receptor whose extracellular amino acid sequence is shown in SEQ ID NO: 9, where the underlined portion represents the signal peptide.

[0054] SEQ ID NO: 9:

[0055] MWSLLLCGLSIALPLSVTA DGCKDIFMKNEILSASQPFAFNCTFPPITSGEV

[0056] SVTWYKNSSKIPVSKIIQSRIHQDETWILFLPMEWGDSGVYQCVIKGRDSC

[0057] HRIHVNLTVFEKHWCDTSIGGLPNLSDEYKQILHLGKDDSLTCHLHFPKS

[0058] CVLGPIKWYKDCNEIKGERFTVLETRLLVSNVSAEDRGNYACQAILTHSG

[0059] KQYEVLNGITVSITERAGYGGSVPKIIYPKNHSIEVQLGTTLIVDCNVTDT

[0060] KDNTNLRCWRVNNTLVDDYYDESKRIREGVETHVSFREHNLYTVNITFL

[0061] EVKMEDYGLPFMCHAGVSTAYIILQLPAPDFR

[0062] In this specification, "anti-human IL-36R monoclonal antibody" means a monoclonal antibody that can bind to human IL-36R with sufficient affinity, such that the monoclonal antibody can be used as a diagnostic and / or therapeutic agent targeting human IL-36R.

[0063] The anti-human IL-36R monoclonal antibody of this application does not bind to proteins unrelated to the target. Here, "unrelated protein" refers to proteins other than human IL-36R, which is the target; and "does not bind" means that, when the binding ability of the anti-human IL-36R monoclonal antibody of this application to human IL-36R, which is its target, is taken as 100%, the binding ability of the anti-human IL-36R monoclonal antibody of this application to the unrelated protein is less than 10%, for example, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0%.

[0064] The anti-human IL-36R monoclonal antibody of this application may not bind to IL-36R from other animal species. Here, "other animal species" refers to animal species other than humans, such as marmosets, cynomolgus monkeys, pigs, dogs, rabbits, rats, mice, guinea pigs, etc. Here, "not binding" means that, when the binding ability of the anti-human IL-36R monoclonal antibody of this application to human IL-36R as its target is taken as 100%, the binding ability of the anti-human IL-36R monoclonal antibody of this application to IL-36R from other animal species is less than 10%, such as 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0%.

[0065] The human IL-36R monoclonal antibody of this application may have an equilibrium dissociation constant (K0) of, for example, ≤1 μM, ≤100 nM, ≤50 nM, or ≤40 nM. D ).

[0066] Experimental results show that the anti-human IL-36R monoclonal antibody of this application can specifically bind to human IL-36R.

[0067] The anti-human IL-36R monoclonal antibody of this application is comparable to or superior to similar commercially available monoclonal antibody products in many aspects of biological activity. These biological activities include, for example, the neutralization of STAT3 phosphorylation induced by human IL-36 (α, β, γ) in cells, the neutralization of CXCL-1 and IL-8 release induced by human IL-36 (α, β, γ) in cells, and the neutralization of IL-8 release induced by human IL-36β in human PBMC cells.

[0068] In one specific embodiment, the amino acid sequence of the heavy chain of the anti-human IL-36R monoclonal antibody of this application may be as shown in SEQ ID NO: 10; the amino acid sequence of the light chain may be as shown in SEQ ID NO: 11.

[0069] SEQ ID NO: 10

[0070] EVQLVESGGGLVQPGGSLRLSCAASGIDLSNYAMGWVRQAPGKGLEWV

[0071] GYISGGGSAYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR

[0072] WAIKSYFFGMDLWGQGTLVTVSSASTKGPSVFPLAPSSKSGGTAALGC

[0073] LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT

[0074] QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP

[0075] KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE

[0076] QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP

[0077] REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT

[0078] TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL

[0079] SPGK

[0080] SEQ ID NO: 11

[0081] DIQMTQSPSSSVSASVGDRVTITCQASEYISSYLAWYQQKPGKAPKLLIYQ

[0082] ASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQTNNAIHTYGGAF

[0083] GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW

[0084] KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT

[0085] HQGLSSPVTKSFNRGEC

[0086] SEQ ID NO: 10 and 11 are both humanized sequences. To eliminate the ADCC and CDC effects of the antibody, the heavy chain constant region underwent an LALA mutation.

[0087] In this specification, "isolated" nucleic acid means a nucleic acid molecule that has been isolated from components of its native environment. Isolated nucleic acid includes nucleic acid molecules that are normally found in cells containing nucleic acid molecules, but which are located outside chromosomes or at chromosomal locations other than their native chromosomal locations.

[0088] In this specification, "isolated nucleic acid encoding anti-IL-36R monoclonal antibody" means one or more nucleic acid molecules encoding the antibody heavy and light chains, including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present at one or more locations in the host cell.

[0089] In this specification, "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid linked to it. This term includes vectors as self-replicating nucleic acid structures as well as vectors integrated into the genome of a host cell into which it has been introduced. Some vectors are capable of directing the expression of nucleic acids operatively linked to them. Such vectors are referred to herein as "expression vectors."

[0090] In this specification, the terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their progeny (regardless of passage number). Progeny may not be identical to parental cells in terms of nucleic acid contents, but may contain mutations. Mutant progeny with the same function or biological activity selected from the initially transformed cells are included in this specification.

[0091] In this specification, "pharmaceutical composition" means an article which is in a form that enables the bioactivity of the active ingredient contained therein to exert its effect, and which does not contain any additional components that would have unacceptable toxicity to the subject to whom the formulation is to be administered.

[0092] In this specification, "pharmaceuticalally acceptable carrier" means a component of the pharmaceutical composition other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0093] In this application, "monoclonal antibody" generally refers to a human antibody, which can be prepared using techniques 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).

[0094] Antibodies can be prepared by administering immunogens to transgenic animals that have been modified to produce intact human antibodies in response to antigen challenge stimulation or to produce intact antibodies with human variable regions. These animals typically contain some or all of the human immunoglobulin loci, which replace endogenous immunoglobulin loci, or are located extrachromosomally or randomly integrated into the animal. In such transgenic mice, endogenous immunoglobulin loci are generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, N., Nat. Biotech. (Nature Biotechnology) 23:1117-1125 (2005). See also, for example, the XENOMOUSE described in U.S. Patent Nos. 6,075,181 and 6,150,584. TM Technology; described in U.S. Patent No. 5,770,429 Technology; described in U.S. Patent No. 7,041,870 The technology described in U.S. Patent Application Publication No. US 2007 / 0061900 The technology allows for the extraction of human variable regions from complete antibodies generated by such animals, for example, through further modification by combining them with different human constant regions.

[0095] Human antibodies can also be prepared using hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma cells used for the production of human monoclonal antibodies have been described (see, for example, Kozbor, D., J. Immunol. 133:3001-3005 (1984); Brodeur, BR et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York (1987), pp. 51-63; Boerner, P. et al., J. Immunol. 147:86-95 (1991)). Human antibodies produced via human B-cell hybridoma technology are also documented 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 generation 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).

[0096] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human phage display libraries, and then such variable domain sequences can be combined with desired human constant domains.

[0097] Human antibodies can also be selected based on autoantibody libraries, meaning that human antibodies can be isolated by screening combinatorial libraries for antibodies with one or more desired activities. For example, various methods for producing phage display libraries and screening such libraries for antibodies with desired binding characteristics are known in the art. These methods are reviewed, for example, in Hoogenboom, HR 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, JD et al., J. Mol. Biol. 222:581-597 (1992); Marks, JD and Bradbury, A., Methods in Molecular Biology. 248:161-175(2003); Sidhu, SS et al., J. Mol. Biol. 338: 299-310 (2004); Lee, CV et 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).

[0098] In some phage display methods, complete sets of VH and VL genes are cloned separately by polymerase chain reaction (PCR), randomly recombined in a phage library, and then antigen-binding phages are screened from the phage library, as described in Winter, G. et al., Ann. Rev. Immunol. 12:433-455 (1994). Phages typically display antibody fragments as single-stranded Fv (scFv) fragments or as Fab fragments. Libraries derived from immunized sources provide high-affinity antibodies against immunogens without the need to construct hybridomas. Alternatively, a non-immunized complete set can be cloned (e.g., from humans) to provide a single source of antibodies against a large number of non-self and self antigens in the absence of any immunization, as described by Griffiths, AD et al., EMBO J, 12:725-734 (1993). Finally, an unimmunized library can also be synthesized by cloning the unrearranged V gene segment from stem cells, encoding a highly variable CDR3 region using PCR primers containing random sequences, and then performing rearrangement in vitro, as described by Hoogenboom, H.R. 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 Publications Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0099] The antibody may also be a multispecific antibody, such as a bispecific antibody. A bispecific antibody is a monoclonal antibody that has binding specificity to at least two different sites. Techniques for generating multispecific antibodies include, but are not limited to, recombinant co-expression of two pairs of immunoglobulin heavy-light chains with different specificities (see Milstein, C. and Cuello, AC, Nature 305:537-540 (1983); WO93 / 08829; and Traunecker, A. et al., EMBO J.10:3655-3659 (1991)) and "segment-entry-cavity" engineering (see, for example, U.S. Patent No. 5,731,168). Engineered electrostatic manipulation effects (WO) can also be used to generate antibody Fc-heterodimeric molecules. 2009 / 089004); crosslinking two or more antibodies or fragments (see, for example, U.S. Patent No. 4,676,980 and Brennan, M. et al., Science 229:81-83 (1985)); using leucine zippers to generate bispecific antibodies (see, for example, Kostelny, SA et al., J. Immunol. 148:1547-1553 (1992)); using “dual antibody” technology for generating bispecific antibody fragments (see, for example, Ho Illiger, P. et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993)); and the use of single-chain Fv (scFv) dimers (see, for example, Gruber, M. et al., J. Immunol. 152: 5368-5374 (1994)); and the preparation of trispecific antibodies (as described, for example, Tutt, A. et al., J. Immunol. 147: 60-69 (1991)) to generate multispecific antibodies.

[0100] The monoclonal antibodies described herein also include engineered antibodies having three or more functional antigen-binding sites, including “octopus antibodies” (see, for example, US 2006 / 0025576).

[0101] The antibodies described herein may also include multispecific antibodies described in WO 2009 / 080251, WO 2009 / 080252, WO2009 / 080253, WO2009 / 080254, WO 2010 / 112193, WO 2010 / 115589, WO2010 / 136172, WO 2010 / 145792, WO2010 / 145793, WO 2011 / 117330, WO 2012 / 025525, WO 2012 / 025530, WO 2013 / 026835, WO2013 / 026831, WO 2013 / 164325, or WO 2013 / 174873.

[0102] The monoclonal antibodies described herein may also be antibody variants, for example, where improved binding affinity and / or other biological properties of the antibody may be desired. Amino acid sequence variants of the antibody can be prepared by introducing suitable modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletion, and / or insertion and / or substitution of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be performed to obtain the final construct, provided that the final construct possesses the desired characteristics, such as antigen binding. Thus, in some embodiments, antibody variants with one or more amino acid substitutions are provided, where the sites of interest for substitution mutation include HVR and FR. For example, amino acid substitutions can be introduced into the antibody of interest and products with desired activities can be screened, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0103] Example

[0104] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0105] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0106] Example 1: Preparation of anti-human IL-36R monoclonal antibody QX009N

[0107] Quanxin Biotechnology developed and expressed human interleukin-36 receptor (hIL-36R-Rabbit Fc), which was used as an immunogen to immunize New Zealand rabbits. B-cell cloning technology was used to obtain antigen-binding specific antibody clones, and then monoclonal antibodies binding to human IL-36R and exhibiting human IL-36R inhibitory activity were screened. The cell supernatant was analyzed and screened using Binding ELISA and HT29 cell IL-8 release methods to select target clones. The entire immunization and screening process was outsourced to a commercial company.

[0108] Ten clones were selected for recombinant expression and sequencing. Clones #25 showed the best cellular activity. Therefore, clone #25 was further humanized. Human IgG germline sequence homology was compared using NCBI IgBlast. IGHV3-66*01 was selected as the heavy chain CDR transplantation template, and the CDR regions of clone 25# heavy chain (i.e., CDR-H1 (SEQ ID No:1), CDR-H2 (SEQ ID No:2), and CDR-H3 (SEQ ID No:3)) were transplanted into the backbone region of IGHV3-66*01. IGKV1-12*01 was selected as the light chain CDR transplantation template, and the CDR regions of clone 25# light chain (i.e., CDR-L1 (SEQ ID No:4), CDR-L2 (SEQ ID No:5), and CDR-L3 (SEQ ID No:6)) were transplanted into the backbone region of IGKV1-12*01. Reversion mutations and site-directed mutations were performed at specific sites in the backbone region and the CDR region to obtain the variable region of the monoclonal antibody QX009N of this application. Finally, the humanized heavy chain variable region sequence is shown in SEQ ID NO: 7; the humanized light chain variable region amino acid sequence is shown in SEQ ID NO: 8.

[0109] The genes for the heavy chain (SEQ ID NO: 10) and the variable region of the light chain (SEQ ID NO: 8) were obtained by PCR amplification. The heavy chain expression plasmid pQX1 was digested with HindIII and BamHI; the light chain expression plasmid pQX2.3 was digested with HindIII and BsiWI; and the PCR-amplified genes were inserted into their respective 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.

[0110] Results of nucleic acid electrophoresis detection of PCR-amplified heavy chain gene fragments, light chain variable region gene fragments, and double-enzyme-digested plasmids are as follows: Figure 1 As shown. According to Figure 1 The results show that the antibody heavy chain and light chain variable region PCR amplification results and the results of double enzyme digestion of heavy chain and light chain expression plasmids are as follows: the heavy chain and light chain plasmids are about 5000bp in size, the heavy chain is about 1469bp, and the light chain variable region is about 441bp.

[0111] The correct heavy chain expression plasmid pQX2.1-25VH-Hu12 (the amino acid sequence of the expressed heavy chain is shown in SEQ ID NO: 10) and the light chain expression plasmid pQX2.3-25VK-Hu17 (the amino acid sequence of the expressed light chain is shown in SEQ ID NO: 11) were co-transfected into ExpiCHO-S cells. One day before transfection, ExpiCHO-S cells were diluted to 3 × 10⁻⁶ cells / mL. 6 Cells were passaged at a density of 10 cells / ml before transfection. On the day of transfection, the cell density was diluted to 6 × 10⁻⁶ cells / ml. 6 Cells / ml, 25ml of cells in a 125ml shake flask, awaiting transfection. The transfection and expression process is as follows: Figure 2 As shown.

[0112] On day 6 post-transfection, the culture supernatant was harvested and purified in one step using Protein A. The purified antibody was detected by SDS-PAGE electrophoresis and named QX009N(HZD25-54). The results of protein electrophoresis for this antibody are as follows: Figure 3 As shown. Protein electrophoresis was performed using a denaturing reducing gel. Figure 3 The results showed two bands, 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).

[0113] Example 2 Equilibrium dissociation constant (K) D Determination of )

[0114] The affinity of QX009N (HZD25-54) for human IL-36R was detected using a Biacore T200 instrument, with all procedures performed at 25°C. A commercial Protein A chip was used, and an appropriate amount of antibody was immobilized using a capture method to achieve an Rmax of approximately 50 RU at a capture flow rate of 10 μl / min. The antigen was serially diluted, and the flow rate was switched to 30 μl / min, flowing sequentially from low to high concentration through the reference channel and the channel containing the immobilized antibody. A buffer solution was used as a negative control. After each binding and dissociation cycle, the chip was regenerated using glycine at pH 1.5. The instrument's built-in analysis software was used to fit the 1:1 binding model in the Kinetics option, and the antibody binding rate constant k was calculated. a dissociation rate constant k d and the dissociation equilibrium constant K D value.

[0115] In addition, the affinity of QX009N (HZD25-54) was compared with that of Spesolimab, a monoclonal antibody against human IL-36R currently in Phase III clinical trials. The detection method for the known antibody was the same as that for QX009N, and the results are shown in Table 1. Spesolimab was prepared by constructing an expression plasmid based on the B6 sequence provided in patent US9023995B2 and transiently transfecting it into ExpiCHO-S cells.

[0116] Table 1. Affinity of anti-human IL-36R monoclonal antibodies to human IL-36R.

[0117] Sample Name <![CDATA[k a (10 5 M -1 S -1 )]]> <![CDATA[k d (10 -5 S -1 )]]> <![CDATA[K D (10 -10 M)]]> QX009N 1.14 3.58 3.13 Spesolimab 0.36 3.11 8.62

[0118] The data in the table is the average value of three tests performed on each sample.

[0119] Example 3: Phosphorylation activity of HT29 reporter gene in cells induced by QX009N (HZD25-54) and Spesolimab analogs and human IL-36 (α, β, γ)

[0120] The activity of QX009N (HZD25-54) antagonizing human IL-36 (α, β, γ) via IL-36R-IL-1RAcp-mediated STAT3 phosphorylation was determined using the HT29 reporter gene cell line: 100 μl of cells per well (40,000 cells) was added to each well of a 96-well cell culture plate and cultured overnight at 37°C and 5% CO2. Antibody was added to the cells and incubated for 1 h, with a final antibody concentration ranging from 0 to 10,000 ng / ml. After incubation, 50 μl / well 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, and the cells were then cultured at 37°C and 5% CO2 for 24 h. Discard the cell culture supernatant, add 120 μl of ONE-Glo-Luciferase Reagent to each well, incubate for 10 min, then transfer 80 μl from each well to a white 96-well plate. Detect the Luminescence signal and plot the dose-response curve to analyze the antibody antagonistic activity. The dose-response curve is shown below. Figure 4 As shown.

[0121] Figure 4 The results showed that QX009N(HZD25-54) could inhibit human IL-36 (α, β, γ)-induced STAT3 phosphorylation of the HT29 reporter gene in cells, and the IC50 of QX009N(HZD25-54) inhibited the activity of human IL-36 (α, β, γ)-induced STAT3 phosphorylation in HT29 reporter gene cells.50 At 2.16 ng / ml, the Spesolimab analogue inhibits the IC50 of human IL-36 (α, β, γ)-induced HT29 reporter gene STAT3 phosphorylation in cells. 50 The concentration was 15.67 ng / ml.

[0122] Example 4: Activity of QX009N (HZD25-54) and Spesolimab analogues in the release of CXCL-1 and IL-8 from HT29 cells induced by human IL-36 (α, β, γ).

[0123] Using the HT29 cell line, the antagonism of human IL-36 (α, β, γ) by QX009N (HZD25-54) was investigated. This antagonism induced the release of CXCL-1 and IL-8 from HT29 cells via IL-36R-IL-1RAcp. 40,000 cells were added to each well of a 96-well cell culture plate at a volume of 100 μl, and the cells were cultured overnight at 37°C and 5% CO2. Antibody was added to the cells and incubated for 1 h, with a final antibody concentration ranging from 0 to 10,000 ng / ml. After incubation, 50 μl / well 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, and the cells were then cultured at 37°C and 5% CO2 for 24 h. Cell culture supernatant was collected, and the expression of CXCL-1 and IL-8 in the supernatant was detected using a sandwich ELISA method. Dose-response curves were then plotted to analyze the antagonistic activity of the antibodies. The dose-response curves are shown below. Figure 5 As shown.

[0124] Figure 5 The results showed that QX009N(HZD25-54) could inhibit the release of CXCL-1 and IL-8 induced by human IL-36 (α, β, γ) from HT29 cells, and the IC50 of QX009N(HZD25-54) in inhibiting the release of CXCL-1 and IL-8 induced by human IL-36 (α, β, γ) from HT29 cells was [not specified]. 50 The IC50 values ​​of Spesolimab analogues, at 2.21 ng / ml and 1.53 ng / ml respectively, inhibited the release of CXCL-1 and IL-8 from HT29 cells induced by human IL-36 (α, β, γ). 50 The concentrations were 12.29 ng / ml and 11.47 ng / ml, respectively.

[0125] Example 5: Activity of QX009N (HZD25-54) and Spesolimab analogues in the release of CXCL-1 and IL-8 from A431 cells induced by human IL-36 (α, β, γ).

[0126] Using the A431 cell line, QX009N (HZD25-54) antagonized human IL-36 (α, β, γ) and induced the release of CXCL-1 and IL-8 from A431 cells via IL-36R-IL-1RAcp: 100 μl of cells per well (40,000 cells) were added to 96-well cell culture plates and cultured overnight at 37°C and 5% CO2. Antibody was added to the cells and incubated for 1 h, with a final antibody concentration ranging from 0 to 10,000 ng / ml. After incubation, 50 μl / well 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, and the cells were then cultured at 37°C and 5% CO2 for 24 h. Cell culture supernatant was collected, and the expression of CXCL-1 and IL-8 in the supernatant was detected using a sandwich ELISA method. Dose-response curves were then plotted to analyze the antagonistic activity of the antibodies. The dose-response curves are shown below. Figure 6 As shown.

[0127] Figure 6 The results showed that QX009N (HZD25-54) could inhibit the release of CXCL-1 and IL-8 induced by human IL-36 (α, β, γ) from A431 cells, and QX009N (HZD25-54) could inhibit the IC50 of the release of CXCL-1 and IL-8 induced by human IL-36 (α, β, γ) from A431 cells. 50 The IC50 values ​​of Spesolimab analogues, at 4.12 ng / ml and 2.89 ng / ml respectively, inhibited the release of CXCL-1 and IL-8 from A431 cells induced by human IL-36 (α, β, γ). 50 The concentrations were 22.46 ng / ml and 16.02 ng / ml, respectively.

[0128] Example 6: Neutralization of the activity of QX009N (HZD25-54) and Spesolimab analogues in inducing IL-8 release from human IL-36β in human PBMC cells.

[0129] The activity of QX009N (HZD25-54) antagonizing human IL-36β via IL-36R-IL-1RAcp-induced IL-8 release from human PBMCs was determined using human PBMCs: PBMCs were isolated from human venous blood (derived from healthy adult volunteers) using density gradient centrifugation. 200,000 cells per well of PBMCs were added to 100 μl of each well in a 96-well cell culture plate. Antibody was added to the PBMCs and incubated for 1 h, with a final antibody concentration ranging from 0 to 5000 ng / ml. After incubation, 50 μl / well of 10 ng / ml recombinant human IL-36β was added, and the plates were then cultured at 37°C and 5% CO2 for 24 h. The cell culture supernatant was collected, and the expression of IL-8 in the supernatant was detected using a sandwich ELISA method. A dose-response curve was plotted to analyze the antagonistic activity of the antibody. The dose-response curve is shown below. Figure 7 As shown.

[0130] from Figure 7 The results showed that QX009N(HZD25-54) could inhibit the release of IL-8 from human PBMCs induced by human IL-36β, and the IC50 of QX009N(HZD25-54) inhibited the release of IL-8 from human PBMCs induced by human IL-36β. 50 The IC50 value of the Spesolimab analogue for inhibiting the release of IL-8 from human IL-36β-induced human PBMCs was 4.69 ng / ml. 50 The concentration was 26.59 ng / ml.

Claims

1. A monoclonal antibody against human interleukin 36 receptor (IL-36R), characterized in that, It includes 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), wherein: The amino acid sequence of CDR-H1 is shown in SEQ ID NO: 1; The amino acid sequence of CDR-H2 is shown in SEQ ID NO: 2; The amino acid sequence of CDR-H3 is shown in SEQ ID NO: 3; The amino acid sequence of CDR-L1 is shown in SEQ ID NO: 4; The amino acid sequence of CDR-L2 is shown in SEQ ID NO: 5; The amino acid sequence of CDR-L3 is shown in SEQ ID NO:

6.

2. The monoclonal antibody according to claim 1, characterized in that, It includes a heavy chain variable region and a light chain variable region, wherein, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7; The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO:

8.

3. An isolated nucleic acid, characterized in that, Encoding the monoclonal antibody according to claim 1 or 2.

4. A host cell, characterized in that, It contains the nucleic acid as described in claim 3.

5. A method for producing monoclonal antibodies, characterized in that, The method includes culturing host cells according to claim 4 to produce monoclonal antibodies according to claim 1 or 2.

6. A pharmaceutical composition, characterized in that, It comprises the monoclonal antibody as described in claim 1 or 2 and a pharmaceutically acceptable vector.

7. Use of the monoclonal antibody according to claim 1 or 2 in the preparation of a medicament for treating IL-36R-mediated signal transduction-related diseases, wherein the IL-36R-mediated signal transduction-related diseases are selected from generalized pustular psoriasis, palmoplantar pustulosis, and chronic obstructive pulmonary disease.

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