Anti-human interleukin 36 receptor monoclonal antibody and use thereof

Abstract

The present invention provides an anti-human interleukin 36 receptor (IL-36R) monoclonal antibody and a use thereof. The 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, and the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are as shown in SEQ ID NO: 1 to SEQ ID NO: 6, respectively. Compared with Spesolimab, the monoclonal antibody has comparable binding affinity to human IL-36R, and the neutralizing activity of the monoclonal antibody at the cellular level is superior to that of Spesolimab.

Description

Anti-human interleukin-36 receptor monoclonal antibody and its application Technical Field

[0001] The present application relates to the field of antibody drugs. Specifically, the present application relates to monoclonal antibodies targeting human interleukin-36 receptor (IL-36R) and their applications. Background Art

[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), IL-36γ (IL1F9), and the IL-36 receptor antagonist IL-36Ra (IL1F5). Its structural pattern is similar to that of the classic IL-1 family: it lacks a signal peptide and cannot be secreted through the classic Golgi-endoplasmic reticulum pathway. It typically forms an inactive precursor that is subsequently activated by proteolysis. IL-36 can be produced by a variety of cell types, including monocytes, macrophages, T / B lymphocytes, keratinocytes, and epithelial cells. The receptor for IL-36 is composed of the specific receptor IL-36R (also known as IL-1RL2, Interleukin-1 receptor-like 2) and the IL-1 receptor accessory protein IL-1RAcP (IL-1 receptor accessory protein), both of which are composed of an extracellular region consisting of three domains, a transmembrane region, and an intracellular Toll / IL-1 receptor (TIR) ​​domain. Proteolytically mature agonist IL-36 ligands (α, β, γ) bind to IL-36R to form a binary complex, which then recruits IL-1RAcP to assemble into a functional ternary complex. The intracellular TIR of IL-36R and IL-1RAcP interacts with each other, aggregates the transduction adaptor protein myeloid differentiation factor 88 (MyD88) and interleukin-1 receptor associated kinase (IRAK), and then activates the downstream NF-κB (Nuclear factor kappa-B) and MAPK (Mitogen-activated protein kinase) signaling pathways. When IL-36 is expressed normally, it can perform host defense through inflammatory responses. When IL-36 expression is dysregulated, it will stimulate its receptor-expressing cells (epithelial cells, fibroblasts, keratinocytes, monocytes, macrophages, dendritic cells, T cells) to produce inflammatory cytokines, chemokines, adhesion molecules, etc., thereby mediating pathological inflammatory responses and participating in the pathological process 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 the pathway leading 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-36 cytokines in homeostasis and pathological conditions[J].Journal of Leukocyte Biology,2015,97(4):645.

[0005] [3]Henry C M,Sullivan G P,Clancy D M,et al.Neutrophil-Derived Proteases Escalate Inflammation through Activation of IL-36 Family Cytokines[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)or Antagonist(IL-36Ra)Activity[J].Journal of Biological Chemistry,2011,286(49):42594-602.

[0007] [5]Buhl,AL,Wenzel,J.Interleukin-36 in Infectious and Inflammatory Skin Diseases[J].Frontiers in Immunology,2019.01162

[0008] Summary of the Invention

[0009] The purpose of the present application is to provide a novel anti-human IL-36R monoclonal antibody, a pharmaceutical composition comprising the monoclonal antibody, and pharmaceutical uses of the monoclonal antibody.

[0010] The technical solution of this application is as follows:

[0011] 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), wherein:

[0012] The amino acid sequence of CDR-H1 (CDR-H1 in this specification represents heavy chain CDR1) is shown in SEQ ID NO: 1 (NYAMG);

[0013] The amino acid sequence of CDR-H2 (CDR-H2 in this specification represents heavy chain CDR2) is shown in SEQ ID NO: 2 (YISGGGSAYYASWAKG);

[0014] The amino acid sequence of CDR-H3 (CDR-H3 in this specification represents heavy chain CDR3) is shown in SEQ ID NO: 3 (WAIKSYFFGMDL);

[0015] The amino acid sequence of CDR-L1 (CDR-L1 in this specification represents light chain CDR1) is shown in SEQ ID NO: 4 (QASEYISSYLA);

[0016] The amino acid sequence of CDR-L2 (CDR-L2 in this specification represents light chain CDR2) is shown in SEQ ID NO: 5 (QASTLAS);

[0017] The amino acid sequence of CDR-L3 (CDR-L3 in this specification represents light chain CDR3) is shown in SEQ ID NO: 6 (QTNNAIHTYGGA).

[0018] 2. The monoclonal antibody according to item 1, comprising a heavy chain variable region and a light chain variable region, wherein:

[0019] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7, which is EVQLVESGGGLVQPGGSLRLSCAASGIDLSNYAMGWVRQAPGKGLEWVGYISGGGSAYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWAIKSYFFGMDLWGQGTLVTVSS;

[0020] The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8, which has an amino acid sequence of DIQMTQSPSSVSASVGDRVTITCQASEYISSYLAWYQQKPGKAPKLLIYQASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQTNNAIHTYGGAFGGGTKVEIK.

[0021] 3. An isolated nucleic acid encoding any one of the aforementioned monoclonal antibodies.

[0022] 4. A host cell comprising the nucleic acid according to item 3.

[0023] 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.

[0024] 5. A method for producing a monoclonal antibody, the method comprising culturing the host cell according to item 4 to thereby produce any one of the aforementioned monoclonal antibodies.

[0025] The method comprises expressing a recombinant vector encoding the anti-human IL-36R monoclonal antibody in a suitable host cell, thereby producing the monoclonal antibody. In certain embodiments, the method comprises culturing a host cell containing a nucleic acid encoding the anti-human IL-36R monoclonal antibody, thereby expressing the nucleic acid. The method may further comprise recovering the anti-human IL-36R monoclonal antibody from the host cell culture or host cell culture medium.

[0026] 6. A pharmaceutical composition comprising any one of the aforementioned monoclonal antibodies and a pharmaceutically acceptable carrier.

[0027] The pharmaceutical composition may further comprise an additional therapeutic agent (eg, a different anti-human IL-36R antibody).

[0028] 7. The pharmaceutical composition according to item 6, which is used to treat diseases related to IL-36R-mediated signal transduction.

[0029] 8. The pharmaceutical composition according to item 7, wherein the IL-36R-mediated signal transduction-related disease is selected from dermatitis, psoriasis, inflammatory bowel disease, arthritis, systemic lupus erythematosus, inflammatory lung disease and chronic kidney disease,

[0030] Preferably, the IL-36R-mediated signal transduction-related disease is 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.

[0031] 9. Use of any of the aforementioned monoclonal antibodies in the preparation of a medicament for treating diseases associated with IL-36R-mediated signal transduction.

[0032] 10. The method according to claim 9, wherein the IL-36R-mediated signal transduction-related disease is selected from dermatitis, psoriasis, inflammatory bowel disease, arthritis, systemic lupus erythematosus, inflammatory lung disease and chronic kidney disease,

[0033] Preferably, the IL-36R-mediated signal transduction-related disease is 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.

[0034] 11. A method for treating a disease associated with IL-36R-mediated signal transduction, comprising:

[0035] The monoclonal antibody according to any one of the preceding items or the pharmaceutical composition according to any one of the preceding items is administered to a subject in need thereof.

[0036] 12. The method according to item 11, wherein the IL-36R-mediated signal transduction-related disease is selected from dermatitis, psoriasis, inflammatory bowel disease, arthritis, systemic lupus erythematosus, inflammatory lung disease and chronic kidney disease,

[0037] Preferably, the IL-36R-mediated signal transduction-related disease is 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.

[0038] The present application provides a new anti-human IL-36R monoclonal antibody, which has a comparable affinity for binding to IL-36R as the anti-human IL-36R monoclonal antibody Spesolimab in the prior art, and has better neutralizing activity at the cellular level than Spesolimab. It should be noted that Spesolimab is a monoclonal antibody drug targeting IL-36R developed by Boehringer Ingelheim. The Phase III clinical trial NAVIGATOR of Spesolimab for the treatment of generalized pustular psoriasis in adults was successful. It has now been approved for marketing by the U.S. Food and Drug Administration and has been granted priority review status by the China National Drug Administration. The monoclonal antibody of the present application (e.g., QX009N (HZD25-54)) can be an IgG1 (immunoglobulin G1) type humanized monoclonal antibody targeting IL-36R (interleukin-36 receptor), has a high affinity for IL-36R, can specifically bind to IL-36R, and block IL-36 inflammatory pathway signals. HZD25-54 binds to IL-36R, competitively blocks the binding of receptor agonists (IL-36α, β, γ) to IL-36R, downregulates downstream pro-inflammatory signaling pathways and pro-fibrotic signaling pathways, inhibits inflammatory responses mediated by epithelial cells / fibroblasts / immune cells, thereby reducing the release of pathogenic cellular inflammatory factors in inflammatory diseases / skin diseases, and achieving the purpose of controlling the disease.

[0039] The monoclonal antibody of the present application shows a neutralizing activity superior to that of Spesolimab (prepared by expression according to the sequence disclosed in the patent) at the cellular level, and is expected to show good clinical effects in preventing and treating related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The accompanying drawings are provided to facilitate a better understanding of the present application and do not constitute an undue limitation on the present application.

[0041] Figure 1 shows the results of nucleic acid electrophoresis for the construction of the HZD25-54 transient expression plasmid. 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.

[0042] Figure 2 is a flowchart of transient expression.

[0043] FIG3 is an electrophoresis detection diagram of QX009N (HZD25-54).

[0044] FIG4 is a graph showing that QX009N (HZD25-54) and Spesolimab analogs neutralize human IL-36 (α, β, γ)-induced STAT3 phosphorylation activity in HT29 reporter cells.

[0045] FIG5 is a graph showing the neutralization of CXCL-1 and IL-8 release from HT29 cells induced by human IL-36 (α, β, γ) by QX009N (HZD25-54) and Spesolimab analogs.

[0046] FIG6 is a graph showing the neutralization of CXCL-1 and IL-8 release from A431 cells induced by human IL-36 (α, β, γ) by QX009N (HZD25-54) and Spesolimab analogs.

[0047] FIG7 is a graph showing that QX009N (HZD25-54) and Spesolimab analogs neutralize the activity of human IL-36β inducing IL-8 release from PBMC cells. DETAILED DESCRIPTION

[0048] The following description of exemplary embodiments of the present application includes various details of the embodiments of the present application to facilitate understanding, and should be considered as merely exemplary. Therefore, those skilled in the art will recognize that various changes and modifications may be made to the embodiments described herein without departing from the scope and spirit of the present application. Similarly, for the sake of clarity and conciseness, descriptions of well-known functions and structures are omitted in the following description.

[0049] The technical terms mentioned in this specification have the same meanings as those generally understood by those skilled in the art. In case of any conflict, the definitions in this specification shall prevail.

[0050] Generally speaking, the terms used in this specification have the following meanings.

[0051] As used herein, an "isolated" antibody is one that has been separated from components of its natural environment. In certain embodiments, the antibody is purified to greater than 95% or 99% purity, as 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. B 848: 79-87 (2007).

[0052] In this specification, " monoclonal antibody " represents the antibody derived from the colony of substantially homologous antibody, that is, each antibody constituting the colony is identical and / or in conjunction with identical epi-position, except possible variant antibody (for example, containing naturally occurring mutation or producing in the production process of monoclonal antibody product), such variant is usually present in trace.Different from the polyclonal antibody product that generally includes the different antibodies for different determinants (epi-positions), every kind of monoclonal antibody of monoclonal antibody product is for the single determinant on antigen.Thus, the modifier " monoclonal " indicates that the antibody derives from the feature of substantially homologous antibody colony, and should not be construed as needing to produce the antibody by any ad hoc method.For example, the monoclonal antibody to be used according to the application can be prepared by various techniques, and the technology includes, but is not limited to hybridoma method, recombinant DNA method, phage display method and the method for using all or part of the transgenic animal comprising human immunoglobulin locus, and such method and other exemplary methods for preparing monoclonal antibody are described herein.

[0053] 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 indicated, "binding affinity" as used in this specification refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the equilibrium dissociation constant (K D Affinity can be measured by common methods known in the art.

[0054] In this specification, 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 region is shown in SEQ ID NO: 9, wherein the underlined portion represents a signal peptide.

[0055] SEQ ID NO: 9:

[0056]

[0057] In this specification, “anti-human IL-36R monoclonal antibody” refers to a monoclonal antibody that can bind to human IL-36R with sufficient affinity so that the monoclonal antibody can be used as a diagnostic agent and / or therapeutic agent targeting human IL-36R.

[0058] The anti-human IL-36R monoclonal antibodies of the present application do not bind to unrelated proteins. Here, "unrelated proteins" refers to proteins other than the target human IL-36R. Here, "does not bind" means that, with the binding ability of the anti-human IL-36R monoclonal antibodies of the present application to the target human IL-36R being 100%, the binding ability of the anti-human IL-36R monoclonal antibodies of the present application to the unrelated proteins is less than 10%, for example, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.

[0059] The anti-human IL-36R monoclonal antibodies of the present 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 macaques, pigs, dogs, rabbits, rats, mice, guinea pigs, etc.; and "not binding" means that, with the binding capacity of the anti-human IL-36R monoclonal antibodies of the present application to its target human IL-36R being taken as 100%, the binding capacity of the anti-human IL-36R monoclonal antibodies of the present application to IL-36R from other animal species is less than 10%, for example, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0%.

[0060] The human IL-36R monoclonal antibody of the present application may have, for example, an equilibrium dissociation constant (K) of ≤1 μM, ≤100 nM, ≤50 nM, or ≤40 nM. D ).

[0061] The experimental results show that the anti-human IL-36R monoclonal antibody of the present application can specifically bind to human IL-36R.

[0062] The anti-human IL-36R monoclonal antibody of the present application is comparable to or superior to similar listed monoclonal antibody products in many biological activities, such as neutralizing the activity of human IL-36 (α, β, γ)-induced STAT3 phosphorylation in cells, neutralizing the activity of human IL-36 (α, β, γ)-induced cell release of CXCL-1 and IL-8, and neutralizing the activity of human IL-36β-induced IL-8 release from human PBMCs.

[0063] In a specific embodiment, the amino acid sequence of the heavy chain of the anti-human IL-36R monoclonal antibody of the present application may be as shown in SEQ ID NO: 10; and the amino acid sequence of the light chain may be as shown in SEQ ID NO: 11.

[0064] SEQ ID NO: 10

[0065]

[0066] SEQ ID NO: 11

[0067]

[0068] SEQ ID NOs: 10 and 11 are both humanized sequences. In order to eliminate the ADCC and CDC effects of the antibody, the heavy chain constant region was subjected to LALA mutation.

[0069] In this specification, an "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from the components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0070] As used herein, "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, including 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.

[0071] As used herein, "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term encompasses vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0072] In this specification, "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include the primary transformed cells and the progeny derived therefrom (regardless of the number of generations). Progeny may not be identical to the parental cell in terms of nucleic acid content, but may contain mutations. Mutant progeny with the same function or biological activity that have been screened or selected for the initially transformed cells are included in this specification.

[0073] In this specification, "pharmaceutical composition" means a preparation that is in a form that allows the biological activity of the active ingredient contained therein to take effect, and that contains no additional components that are unacceptably toxic to the subject to which the formulation is to be administered.

[0074] In this specification, "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition other than the active ingredient, which is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0075] In this application, "monoclonal antibody" is generally 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).

[0076] Antibodies can be prepared by administering immunogens to transgenic animals that have been modified to stimulate the production of complete human antibodies or complete antibodies with human variable regions in response to antigenic attack. These animals typically contain a portion or all of the human immunoglobulin loci that replace the endogenous immunoglobulin loci, or are present outside the chromosomes or randomly integrated into the animal. In such transgenic mice, the endogenous immunoglobulin loci are generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, N., Nat. Biotech. 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; US Patent No. 5,770,429 describes Technology; US Patent No. 7,041,870 describes Technology, and U.S. Patent Application Publication No. US 2007 / 0061900 Human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

[0077] Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma cells for producing 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 described in Li, J. et al., Proc. Natl. Acad. Sci. USA 103: 3557-3562 (2006). Other methods include those described in, for example, U.S. Pat. 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).

[0078] Human antibodies can also be produced by isolating Fv clone variable domain sequences selected from human-derived phage display libraries, and these variable domain sequences can then be combined with the desired human constant domains.

[0079] Human antibodies can also be selected based on self-antibody libraries, that is, human antibodies can be isolated by screening combinatorial libraries for antibodies with desired one or more activities. For example, a variety of methods for producing phage display libraries and screening such libraries for antibodies with desired binding characteristics are known in the art. This method is reviewed in, for example, Hoogenboom, HR et al., Methods in Molecular Biology 178: 1-37 (2001), and is further described in, for example, 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).

[0080] In certain phage display methods, repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, which is then screened for antigen-binding phage, as described in Winter, G. et al., Ann. Rev. Immunol. 12: 433-455 (1994). Phage typically display antibody fragments as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, non-immunized repertoires can be cloned (e.g., from humans) to provide a single source of antibodies to a large number of non-self and also self-antigens in the absence of any immunization, as described by Griffiths, AD et al., EMBO J, 12: 725-734 (1993). Finally, unimmunized libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and achieve rearrangement in vitro, as described by Hoogenboom, HR 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.

[0081] The antibody can also be a multispecific antibody, such as a bispecific antibody. Bispecific antibodies are monoclonal antibodies with binding specificity to at least two different sites. The technology for generating multispecific antibodies includes but is not limited to the recombinant co-expression of two pairs of immunoglobulin heavy chain-light chains with different specificities (see Milstein, C. and Cuello, AC, Nature 305: 537-540 (1983); WO 93 / 08829; and Traunecker, A. et al., EMBO J. 10: 3655-3659 (1991)) and "node-into-hole" engineering (see, for example, U.S. Patent No. 5,731,168). It can also be achieved by engineering electrostatic manipulation effects for generating antibody Fc-heterodimer molecules (WO 2009 / 089004); cross-linking two or more antibodies or fragments (see, e.g., 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, e.g., Kostelny, SA et al., J. Immunol. 148:1547-1553 (1992)); using "diabody" technology for generating bispecific antibody fragments (see, e.g., Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (see, e.g., Gruber, M. et al., J. Immunol. 152:5368-5374 (1994)); and preparing trispecific antibodies (as described, e.g., in Tutt, A. et al., J. Immunol. 147:60-69 (1991)) to generate multispecific antibodies.

[0082] The monoclonal antibodies described herein also include engineered antibodies with three or more functional antigen-binding sites, including "octopus antibodies" (see, eg, US 2006 / 0025576).

[0083] The antibodies herein may also include the multispecific antibodies described in WO 2009 / 080251, WO 2009 / 080252, WO 2009 / 080253, WO 2009 / 080254, WO 2010 / 112193, WO 2010 / 115589, WO 2010 / 136172, WO 2010 / 145792, and WO 2010 / 145793, WO 2011 / 117330, WO 2012 / 025525, WO 2012 / 025530, WO 2013 / 026835, WO 2013 / 026831, WO 2013 / 164325, or WO 2013 / 174873.

[0084] The monoclonal antibodies described herein may also be antibody variants, for example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Suitable modifications may be introduced into the nucleotide sequence encoding the antibody, or amino acid sequence variants of the antibody may be prepared by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions may be performed to obtain the final construct, as long as the final construct has the desired characteristics, such as antigen binding. Therefore, in certain embodiments, antibody variants with one or more amino acid substitutions are provided, and the sites of interest for substitution mutations include HVR and FR, for example, amino acid substitutions can be introduced into the antibody of interest and screened for products with the desired activity, for example, retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0085] Example

[0086] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0087] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0088] Example 1 Preparation of anti-human IL-36R monoclonal antibody QX009N

[0089] Quanxin Biotech used its own expressed human interleukin-36 receptor (hIL-36R-Rabbit Fc) as an immunogen to immunize New Zealand rabbits. Using B cell cloning technology, they obtained antigen-binding specific antibody clones, which they then screened for monoclonal antibodies that bind to and inhibit human IL-36R. Cell supernatants were analyzed and screened using binding ELISA and HT29 cell IL-8 release assays to select target clones. The immunization and screening processes were outsourced to a commercial company.

[0090] Ten clones were selected for recombinant expression and sequencing. Clone #25 demonstrated the highest neutralizing activity in cells. Therefore, clone #25 was humanized. Human IgG germline sequence (Germline) homology alignment was performed using NCBI IgBlast, and IGHV3-66*01 was selected as the heavy chain CDR transplantation template. The CDR region of the 25# clone heavy chain (i.e., CDR-H1 (SEQ ID No: 1), CDR-H2 (SEQ ID No: 2), and CDR-H3 (SEQ ID No: 3)) was transplanted into the framework region of IGHV3-66*01; IGKV1-12*01 was selected as the light chain CDR transplantation template, and the CDR region of the 25# clone light chain (i.e., CDR-L1 (SEQ ID No: 4), CDR-L2 (SEQ ID No: 5), and CDR-L3 (SEQ ID No: 6)) was transplanted into the framework region of IGKV1-12*01; back mutations were performed on specific sites in the framework region and site-directed mutagenesis of the CDR region to obtain the variable region 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; the amino acid sequence of the humanized light chain variable region is shown in SEQ ID NO: 8.

[0091] The heavy chain gene (SEQ ID NO: 10) and the light chain variable region gene (SEQ ID NO: 8) were amplified by PCR. 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 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.

[0092] The results of nucleic acid electrophoresis detection of the PCR-amplified heavy chain gene fragment, light chain variable region gene fragment, and double-enzyme digested plasmid are shown in Figure 1. According to the results in Figure 1, the PCR amplification results of the antibody heavy chain and light chain variable regions and the double-enzyme digested heavy chain and light chain expression plasmids show that the plasmid size of the heavy chain and light chain is approximately 5000 bp, the heavy chain is approximately 1469 bp, and the light chain variable region is approximately 441 bp.

[0093] The 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) with the correct sequence were co-transfected into ExpiCHO-S cells. One day before transfection, ExpiCHO-S cells were diluted to 3×10 6 On the day of transfection, the cell density was diluted to 6×10 6 cells / ml, 25ml of cells were placed in a 125ml shake flask and ready for transfection. The transfection and expression process is shown in Figure 2.

[0094] On day 6 after transfection, the culture supernatant was harvested and purified using Protein A. The purified antibody, designated QX009N (HZD25-54), was analyzed by SDS-PAGE electrophoresis. The results of protein electrophoresis for this antibody are shown in Figure 3. Protein electrophoresis was performed on a denaturing reducing gel. Figure 3 shows two bands, approximately 50 kDa and 25 kDa, respectively, which are consistent with the theoretical molecular weights of the heavy chain (49.0 kDa) and light chain (23.4 kDa).

[0095] Example 2 Equilibrium dissociation constant (K D )

[0096] Biacore T200 was used to detect the affinity of QX009N (HZD25-54) to human IL-36R, and all processes were carried out at 25°C. A commercial Protein A chip was used, and an appropriate amount of antibody was fixed by the capture method so that Rmax was around 50RU, and the capture flow rate was 10μl / min. The antigen was gradient diluted, and the instrument flow rate was switched to 30μl / min. The flow was passed through the reference channel and the channel of the fixed antibody in order from low to high concentration, and the buffer solution was used as a negative control. After each binding and dissociation was completed, the chip was regenerated with pH1.5 glycine. The 1:1 binding model in the Kinetics option was selected in the analysis software provided by the instrument for fitting, and the binding rate constant k of the antibody was calculated. a , the dissociation rate constant k d and the dissociation equilibrium constant K D value.

[0097] In addition, the affinity of QX009N (HZD25-54) was compared with that of Spesolimab, a monoclonal antibody targeting human IL-36R currently in Phase III clinical trials. The detection method for the known antibody was the same as that used for QX009N, and the results are shown in Table 1. 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.

[0098] Table 1 Affinity of anti-human IL-36R monoclonal antibodies binding to human IL-36R

[0099] Sample name k a (10 5 M -1 S -1 )k d (10 -5 S -1 )K D (10 -10 M)QX009N1.143.583.13Spesolimab0.363.118.62

[0100] The data in the table are: each sample was tested three times and the average value was calculated.

[0101] Example 3 QX009N (HZD25-54) and Spesolimab analogs neutralize human IL-36 (α, β, γ)-induced STAT3 phosphorylation activity in HT29 reporter cells

[0102] The HT29 reporter cell line was used to determine the activity of QX009N (HZD25-54) in antagonizing human IL-36 (α, β, γ) through IL-36R-IL-1RAcp-mediated phosphorylation of the intracellular signaling molecule STAT3: 40,000 cells were added to a 96-well cell culture plate at a volume of 100 μl per well and incubated overnight at 37°C and 5% CO2. Antibodies were added to the cells for 1 hour, with final antibody concentrations 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 incubated at 37°C and 5% CO2 for 24 hours. The cell culture supernatant was discarded, and 120 μl of ONE-Glo-Luciferase Reagent was added to each well for 10 min. 80 μl of the culture medium was transferred from each well to a white 96-well plate. The luminescence fluorescence signal value was detected and a dose-effect curve was drawn to analyze the antagonistic activity of the antibody. The dose-effect curve is shown in Figure 4.

[0103] The results shown in Figure 4 show that QX009N (HZD25-54) can inhibit the phosphorylation of STAT3 induced by human IL-36 (α, β, γ) in HT29 reporter gene cells. The IC values ​​of QX009N (HZD25-54) for inhibiting the phosphorylation of STAT3 induced by human IL-36 (α, β, γ) in HT29 reporter gene cells are 0. 50 The IC of Spesolimab analogs for inhibiting STAT3 phosphorylation induced by human IL-36 (α, β, γ) in HT29 reporter gene cells was 2.16 ng / ml. 50 It is 15.67ng / ml.

[0104] Example 4: QX009N (HZD25-54) and Spesolimab analogs neutralize the activity of CXCL-1 and IL-8 release induced by human IL-36 (α, β, γ) in HT29 cells

[0105] The HT29 cell line was used to determine whether QX009N (HZD25-54) antagonized human IL-36 (α, β, γ) by inducing CXCL-1 and IL-8 release from HT29 cells via IL-36R-IL-1RAcp: 40,000 cells were plated in 96-well cell culture plates at 100 μl per well and incubated overnight at 37°C and 5% CO2. Antibodies were added to the cells for 1 hour, with final antibody concentrations 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 incubated at 37°C and 5% CO2 for 24 hours. The cell culture supernatant was collected, and the expression of CXCL-1 and IL-8 in the supernatant was detected by sandwich ELISA. A dose-effect curve was drawn to analyze the antagonistic activity of the antibody. The dose-effect curve is shown in Figure 5.

[0106] The results shown in Figure 5 show that QX009N (HZD25-54) can inhibit the release of CXCL-1 and IL-8 induced by human IL-36 (α, β, γ) from HT29 cells. The IC values ​​of QX009N (HZD25-54) for inhibiting the release of CXCL-1 and IL-8 induced by human IL-36 (α, β, γ) from HT29 cells are 0. 50 The IC values ​​of Spesolimab analogs for inhibiting the release of CXCL-1 and IL-8 by HT29 cells induced by human IL-36 (α, β, γ) were 2.21 ng / ml and 1.53 ng / ml, respectively. 50 They are 12.29ng / ml and 11.47ng / ml respectively.

[0107] Example 5: QX009N (HZD25-54) and Spesolimab analogs neutralize the activity of A431 cells induced by human IL-36 (α, β, γ) to release CXCL-1 and IL-8

[0108] The A431 cell line was used to test the effect of QX009N (HZD25-54) on the antagonism of human IL-36 (α, β, γ) by A431 cells via the IL-36R-IL-1RAcp: 40,000 cells were added to a 96-well cell culture plate at a volume of 100 μl per well and incubated overnight at 37°C and 5% CO2. Antibodies were added to the cells for 1 hour, with final antibody concentrations 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 incubated at 37°C and 5% CO2 for 24 hours. The cell culture supernatant was collected, and the expression of CXCL-1 and IL-8 in the supernatant was detected by sandwich ELISA. A dose-effect curve was drawn to analyze the antagonistic activity of the antibody. The dose-effect curve is shown in Figure 6.

[0109] The results shown in Figure 6 show that 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 QX009N (HZD25-54) can inhibit the release of CXCL-1 and IL-8 from A431 cells induced by human IL-36 (α, β, γ). 50 The IC values ​​of Spesolimab analogs for inhibiting the release of CXCL-1 and IL-8 by A431 cells induced by human IL-36 (α, β, γ) were 4.12 ng / ml and 2.89 ng / ml, respectively. 50 They are 22.46ng / ml and 16.02ng / ml respectively.

[0110] Example 6 QX009N (HZD25-54) and Spesolimab analogs neutralize the activity of human IL-36β in inducing IL-8 release from human PBMC cells

[0111] Human PBMC cells were used to determine the activity of QX009N (HZD25-54) in antagonizing human IL-36β and inducing IL-8 release from human PBMC cells through IL-36R-IL-1RAcp: PBMCs were separated from human venous blood (from healthy adult volunteers) by density gradient centrifugation, and 200,000 cells of PBMCs were added to a 96-well cell culture plate at 100 μl per well. Antibodies were added to the PBMCs and incubated for 1 hour, where the final concentration of the antibodies ranged from 0 to 5000 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 hours. The cell culture supernatant was collected, and the expression of IL-8 in the supernatant was detected by sandwich ELISA. A dose-effect curve was drawn to analyze the antagonistic activity of the antibody, and the dose-effect curve is shown in Figure 7.

[0112] The results shown in Figure 7 show that QX009N (HZD25-54) can inhibit the release of IL-8 from human PBMC cells induced by human IL-36β. The IC value of QX009N (HZD25-54) for inhibiting the release of IL-8 from human PBMC cells induced by human IL-36β is 2. 50 The IC value of Spesolimab analogs for inhibiting the release of IL-8 from human PBMC induced by human IL-36β is 4.69 ng / ml. 50 It is 26.59ng / ml.

Claims

1. An anti-human interleukin-36 receptor (IL-36R) monoclonal antibody, characterized in that It 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: 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 comprising 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 light chain variable region is shown in SEQ ID NO:

8.

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

4. A host cell, characterized in that Comprising the nucleic acid according to claim 3.

5. A method for producing a monoclonal antibody, characterized in that: The method comprises culturing the host cell according to claim 4 to produce the monoclonal antibody according to claim 1 or 2.

6. A pharmaceutical composition, characterized in that Comprising the monoclonal antibody according to claim 1 or 2 and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, characterized in that Used to treat IL-36R-mediated signal transduction related diseases.

8. The pharmaceutical composition according to claim 7, characterized in that The IL-36R-mediated signal transduction-related disease is selected from dermatitis, psoriasis, inflammatory bowel disease, arthritis, systemic lupus erythematosus, inflammatory lung disease and chronic kidney disease, Preferably, the IL-36R-mediated signal transduction-related disease is 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.

9. Use of the monoclonal antibody according to claim 1 or 2 in the preparation of a medicament for treating diseases related to IL-36R-mediated signal transduction.

10. The use according to claim 9, characterized in that The IL-36R-mediated signal transduction-related disease is selected from dermatitis, psoriasis, inflammatory bowel disease, arthritis, systemic lupus erythematosus, inflammatory lung disease and chronic kidney disease, Preferably, the IL-36R-mediated signal transduction-related disease is 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 and ankylosing spondylitis.