WO2024082383A1 - 抗人白介素36受体单克隆抗体及其应用 - Google Patents

抗人白介素36受体单克隆抗体及其应用 Download PDF

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WO2024082383A1
WO2024082383A1 PCT/CN2022/134732 CN2022134732W WO2024082383A1 WO 2024082383 A1 WO2024082383 A1 WO 2024082383A1 CN 2022134732 W CN2022134732 W CN 2022134732W WO 2024082383 A1 WO2024082383 A1 WO 2024082383A1
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cdr
monoclonal antibody
human
seq
disease
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French (fr)
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裘霁宛
李望
周毅
陈卫
孔永�
乔怀耀
吴亦亮
陈涛
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江苏荃信生物医药股份有限公司
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  • the present application relates to the field of antibody drugs. Specifically, the present application relates to monoclonal antibodies against human interleukin 36 receptor (IL-36R) and their applications.
  • IL-36R human interleukin 36 receptor
  • Interleukin-36 belongs to the IL-1 family (IL-1 family, IL1F), and is composed of IL-36 receptor agonists IL-36 ⁇ (IL1F6), IL-36 ⁇ (IL1F8), IL-36 ⁇ (IL1F9) and IL-36 receptor antagonist IL-36Ra (IL-36 receptor antagonist, 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 usually forms an inactive precursor first and then is activated by protease hydrolysis.
  • IL-36 can be produced by a variety of cells, including monocytes, macrophages, T/B lymphocytes, keratinocytes, and epithelial cells.
  • the receptor of 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 auxiliary protein IL-1RAcP (IL-1 receptor accessory protein), both of which are composed of an extracellular region composed of three domains, a transmembrane region, and an intracellular Toll/IL-1 receptor (Toll/IL-1 receptor, TIR) domain.
  • IL-1RL2 also known as IL-1RL2, Interleukin-1 receptor-like 2
  • IL-1 receptor auxiliary protein IL-1RAcP IL-1 receptor accessory protein
  • IL-36 ligands 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 interact with each other, aggregate the transduction adaptor protein myeloid differentiation factor 88 (MyD88) and interleukin-1 receptor associated kinase (IRAK), and then activate the downstream NF- ⁇ B (Nuclear factor kappa-B) and MAPK (Mitogen-activated protein kinase) signaling pathways.
  • MyD88 transduction adaptor protein myeloid differentiation factor 88
  • IRAK interleukin-1 receptor associated kinase
  • NF- ⁇ B Nuclear factor kappa-B
  • MAPK Mitogen-activated protein kinase
  • IL-36 expression 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.
  • receptor-expressing cells epidermatitis, fibroblasts, keratinocytes, monocytes, macrophages, dendritic cells, T cells
  • pathological inflammatory responses such as generalized pustular psoriasis, palmoplantar pustulosis, atopic dermatitis, inflammatory bowel disease, and chronic obstructive pulmonary disease.
  • 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 the pharmaceutical use of the monoclonal antibody.
  • An anti-human IL-36R monoclonal antibody comprising three heavy chain complementary determining regions (CDR-H1, CDR-H2 and CDR-H3) and three light chain complementary determining regions (CDR-L1, CDR-L2 and CDR-L3), wherein:
  • CDR-H1 represents heavy chain CDR1
  • SEQ ID NO: 1 NAAMG
  • CDR-H2 represents heavy chain CDR2
  • SEQ ID NO: 2 YISGGGSAYYASWAKG
  • CDR-H3 represents heavy chain CDR3
  • SEQ ID NO: 3 WAIKSYFFGMDL
  • CDR-L1 represents light chain CDR1
  • SEQ ID NO: 4 QASEYISSYLA
  • CDR-L2 represents light chain CDR2
  • SEQ ID NO: 5 QASTLAS
  • CDR-L3 represents light chain CDR3
  • SEQ ID NO: 6 QTNNAIHTYGGA
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7, and its amino acid sequence is EVQLVESGGGLVQPGGSLRLSCAASGIDLSNYAMGWVRQAPGKGLEWVGYISGGGSAYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWAIKSYFFGMDLWGQGTLVTVSS;
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8, and its amino acid sequence is DIQMTQSPSSVSASVGDRVTITCQASEYISSYLAWYQQKPGKAPKLLIYQASTLASGVPSRFSGSGTDFTLTISSLQPEDFATYYCQTNNAIHTYGGAFGGGTKVEIK.
  • a host cell comprising the nucleic acid according to item 3.
  • the nucleic acid may be present on a vector.
  • the vector may be of any type, for example, a recombinant vector such as an expression vector. Any of a variety of host cells may be used.
  • the host cell is a prokaryotic cell, for example, Escherichia coli (E. coli).
  • the host cell is a eukaryotic cell, for example, a mammalian cell, such as a Chinese hamster ovary (CHO) cell.
  • a method for producing a monoclonal antibody comprising culturing the host cell according to item 4 to produce any one of the aforementioned monoclonal antibodies.
  • 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.
  • the method comprises culturing a host cell comprising 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 the host cell culture medium.
  • a pharmaceutical composition comprising any one of the aforementioned monoclonal antibodies and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may further comprise an additional therapeutic agent (e.g., a different anti-human IL-36R antibody).
  • an additional therapeutic agent e.g., a different anti-human IL-36R antibody.
  • 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,
  • 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.
  • 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,
  • 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.
  • a method for treating a disease associated with IL-36R-mediated signal transduction comprising:
  • the monoclonal antibody according to any of the preceding items or the pharmaceutical composition according to any of the preceding items is administered to a subject in need thereof.
  • 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,
  • 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.
  • the present application provides a new anti-human IL-36R monoclonal antibody, which has a comparable affinity for binding to IL-36R compared to the anti-human IL-36R monoclonal antibody Spesolimab in the prior art, and has a better neutralization activity at the cellular level than Spesolimab.
  • 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 been approved for marketing by the U.S. Food and Drug Administration and has obtained priority review qualifications from the China National Drug Administration.
  • the monoclonal antibody of the present application can be a humanized monoclonal antibody of the IgG1 (immunoglobulin G1) type 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.
  • IgG1 immunoglobulin G1
  • IL-36R interleukin-36 receptor
  • HZD25-54 binds to IL-36R, competitively blocks the binding of receptor agonists (IL-36 ⁇ , ⁇ , ⁇ ) 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 cellular inflammatory factors that drive pathogenicity in inflammatory diseases/skin diseases, and achieves the purpose of controlling the disease.
  • receptor agonists IL-36 ⁇ , ⁇ , ⁇
  • the monoclonal antibody of the present application shows a neutralizing activity superior to that of Spesolimab (prepared and expressed according to the sequence disclosed in the patent) at the cellular level, and is expected to show good clinical effects in the prevention and treatment of related diseases.
  • Figure 1 is a diagram showing the results of nucleic acid electrophoresis for constructing the HZD25-54 transient expression plasmid, wherein 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.
  • FIG2 is a flowchart of transient expression.
  • FIG. 3 is an electrophoresis detection diagram of QX009N (HZD25-54).
  • FIG. 4 is a graph showing that QX009N (HZD25-54) and Spesolimab analogs neutralize human IL-36 ( ⁇ , ⁇ , ⁇ )-induced STAT3 phosphorylation activity in HT29 reporter cells.
  • FIG. 5 is a graph showing the activity of QX009N (HZD25-54) and Spesolimab analogs in neutralizing the release of CXCL-1 and IL-8 from HT29 cells induced by human IL-36 ( ⁇ , ⁇ , ⁇ ).
  • FIG. 6 is a graph showing the activity of QX009N (HZD25-54) and Spesolimab analogs in neutralizing the release of CXCL-1 and IL-8 from A431 cells induced by human IL-36 ( ⁇ , ⁇ , ⁇ ).
  • FIG. 7 is a graph showing that QX009N (HZD25-54) and Spesolimab analogs neutralize the activity of human IL-36 ⁇ inducing IL-8 release from PBMC cells.
  • an “isolated” antibody is one that has been separated from the components of its natural environment.
  • 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).
  • electrophoresis e.g., SDS-PAGE isoelectric focusing (IEF), capillary electrophoresis
  • chromatography e.g., ion exchange or reversed phase HPLC.
  • monoclonal antibody means an antibody derived from a population of substantially homologous antibodies, that is, each antibody constituting the population is identical and/or binds to the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or produced during the production of monoclonal antibody products), such variants are usually present in trace amounts.
  • polyclonal antibody products that generally include different antibodies for different determinants (epitopes)
  • each monoclonal antibody of a monoclonal antibody product is directed to a single determinant on an antigen.
  • the modifier "monoclonal” indicates that the antibody is derived from a substantially homologous antibody population, and should not be interpreted as requiring the antibody to be produced by any ad hoc method.
  • the monoclonal antibody to be used according to the present 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 comprising all or part of human immunoglobulin loci, and such methods and other exemplary methods for preparing monoclonal antibodies are described herein.
  • affinity refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen).
  • binding affinity refers to the intrinsic binding affinity reflecting 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 represented by an equilibrium dissociation constant ( KD ). Affinity can be measured by common methods known in the art.
  • human interleukin-36 receptor refers to a membrane receptor derived from humans, whose extracellular domain amino acid sequence is shown in SEQ ID NO: 9, wherein the underlined portion represents the signal peptide.
  • anti-human IL-36R monoclonal antibody means 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.
  • the anti-human IL-36R monoclonal antibody of the present application does not bind to proteins that are unrelated to the target.
  • unrelated proteins refer to proteins other than human IL-36R as the target; here, “no binding” means: when the binding ability of the anti-human IL-36R monoclonal antibody of the present application to human IL-36R as its target is taken as 100%, the binding ability of the anti-human IL-36R monoclonal antibody of the present application to the unrelated protein is less than 10%, for example, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.
  • the anti-human IL-36R monoclonal antibody of the present application may not bind to IL-36R of other animal species.
  • other animal species refers to animal species other than humans, such as marmosets, crab-eating macaques, pigs, dogs, rabbits, rats, mice, guinea pigs, etc.; here, "no binding” means: when the binding ability of the anti-human IL-36R monoclonal antibody of the present application to human IL-36R as its target is taken as 100%, the binding ability of the anti-human IL-36R monoclonal antibody of the present application to IL-36R of other animal species is less than 10%, such as 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.
  • the human IL-36R monoclonal antibody of the present application may have an equilibrium dissociation constant (K D ) of, for example, ⁇ 1 ⁇ M, ⁇ 100 nM, ⁇ 50 nM, or ⁇ 40 nM.
  • K D equilibrium dissociation constant
  • the experimental results show that the anti-human IL-36R monoclonal antibody of the present application can specifically bind to human IL-36R.
  • the anti-human IL-36R monoclonal antibody of the present application is comparable to or superior to similar monoclonal antibody products on the market in many biological activities, such as neutralizing the activity of STAT3 phosphorylation induced by human IL-36 ( ⁇ , ⁇ , ⁇ ) in cells, neutralizing the activity of CXCL-1 and IL-8 induced by human IL-36 ( ⁇ , ⁇ , ⁇ ) in cells, and neutralizing the activity of IL-8 induced by human IL-36 ⁇ in human PBMC cells.
  • amino acid sequence of the heavy chain of the anti-human IL-36R monoclonal antibody of the present application can be as shown in SEQ ID NO: 10; the amino acid sequence of the light chain can be as shown in SEQ ID NO: 11.
  • SEQ ID NO: 10 and 11 are both humanized sequences.
  • the heavy chain constant region was mutated by LALA.
  • an "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains 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.
  • 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.
  • vector means a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which it has 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.”
  • host cell refers to cells into which exogenous nucleic acids have been introduced, including the offspring of such cells.
  • Host cells include “transformants” and “transformed cells”, which include the primary transformed cells and the offspring derived therefrom (regardless of the number of generations).
  • Offspring may not be completely identical to the parental cells in terms of nucleic acid content, but may contain mutations. Mutant offspring with the same function or biological activity that are screened or selected for the initially transformed cells are included in this specification.
  • pharmaceutical composition means a preparation which is in a form that enables the biological activity of the active ingredient contained therein to exert its effect and which does not contain additional components that are unacceptably toxic to the subject to which the preparation is to be administered.
  • pharmaceutically acceptable carrier refers to an ingredient in a pharmaceutical composition other than an active ingredient, which is non-toxic to a subject.
  • Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.
  • “monoclonal antibody” generally refers to human antibodies, which can be prepared using techniques known to those skilled in the art.
  • human antibodies are generally described in van Dijk, M.A. and van de Winkel, J.G., Curr. Opin. Pharmacol. 5:368-374 (2001) and Lonberg, N., Curr. Opin. Immunol. 20:450-459 (2008).
  • 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 challenge. These animals typically contain a portion or all of the human immunoglobulin loci, which 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.
  • transgenic animals see Lonberg, N., Nat. Biotech. (Nature Biotechnology) 23: 1117-1125 (2005). See also, for example, the XENOMOUSE TM technology described in U.S. Pat. Nos.
  • 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, e.g., Kozbor, D., J. Immunol. 133: 3001-3005 (1984); Brodeur, B. R. et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York (1987), pp. 51-63; Boerner, P. et al., 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.
  • Human antibodies can also be made by isolating Fv clone variable domain sequences selected from human-derived phage display libraries, and such variable domain sequences can then be combined with the desired human constant domains.
  • Human antibodies can also be selected based on antibody libraries, that is, human antibodies can be isolated by screening combinatorial libraries for antibodies with the desired one or more activities. For example, a variety of methods for producing phage display libraries and screening such libraries for antibodies with the desired binding characteristics are known in the art. This method is reviewed in, for example, Hoogenboom, H.R. 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, J.D. et al., J. Mol. Biol.
  • phage display methods the 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). Phages typically display antibody fragments as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to immunogens without the need to construct hybridomas.
  • PCR polymerase chain reaction
  • 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 without any immunization, as described by Griffiths, A.D. et al., EMBO J, 12: 725-734 (1993).
  • 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, H.R. and Winter, G., J. Mol. Biol. 227:381-388 (1992).
  • Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373 and U.S. Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936 and 2009/0002360.
  • the antibody may also be a multispecific antibody, such as a bispecific antibody.
  • a bispecific antibody is a monoclonal antibody with 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 chain-light chains with different specificities (see Milstein, C. and Cuello, A.C., 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, e.g., U.S. Pat. No. 5,731,168).
  • antibody Fc-heterodimer molecules by engineering electrostatic manipulation effects (WO 2009/089004); cross-link 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)); use leucine zippers to generate bispecific antibodies (see, for example, Kostelny, S.A. et al., J. Immunol. 148:1547-1553 (1992)); use for generating bispecific antibodies
  • the "diabody” technology of antibody fragments see, for example, Holliger, P. et al., Proc. Natl. Acad. Sci.
  • the monoclonal antibodies described herein also include engineered antibodies having three or more functional antigen binding sites, including "octopus antibodies” (see, e.g., US 2006/0025576).
  • 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, WO2013/026831, WO 2013/164325, or WO 2013/174873.
  • 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.
  • the amino acid sequence variants of the antibody may 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 in the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution may be performed to obtain the final construct, as long as the final construct has the desired characteristics, such as antigen binding.
  • 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 may be introduced into the antibody of interest and screened for products with desired activity, for example, retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.
  • Quanxin Biotech used the self-expressed human interleukin 36 receptor (hIL-36R-Rabbit Fc) as an immunogen to immunize New Zealand rabbits, and used B cell cloning technology to obtain antigen-binding specific antibody clones, and then screened out monoclonal antibodies that bind to human IL-36R and have human IL-36R inhibitory activity.
  • the cell supernatant was analyzed and screened by Binding ELISA and HT29 cell release of IL-8 to select the target clone.
  • the above immunization and screening processes were entrusted to commercial companies.
  • clone 25 was humanized. NCBI IgBlast was used to perform homology alignment of human IgG germline sequences, and IGHV3-66*01 was selected as the heavy chain CDR transplantation template, and 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
  • the heavy chain gene (SEQ ID NO: 10) and the light chain variable region gene (SEQ ID NO: 8) were obtained by PCR amplification.
  • the heavy chain expression plasmid pQX1 was double-digested with HindIII and BamHI; 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.
  • the results of nucleic acid electrophoresis detection of the heavy chain gene fragment amplified by PCR, the light chain variable region gene fragment and the double enzyme digestion plasmid are shown in Figure 1. According to the results in Figure 1, it can be seen that the antibody heavy chain and light chain variable region PCR amplification results and the double enzyme digestion results of the heavy chain and light chain expression plasmids, among which the plasmid size of the heavy chain and light chain is about 5000bp, the heavy chain is about 1469bp, and the light chain variable region is about 441bp.
  • 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.
  • ExpiCHO-S cells were diluted to 3 ⁇ 10 6 cells/ml for pre-transfection passage.
  • the cell density was diluted to 6 ⁇ 10 6 cells/ml, and 25 ml of cells were placed in a 125 ml shake flask and waited for transfection.
  • the transfection and expression process is shown in Figure 2.
  • the affinity of QX009N (HZD25-54) to human IL-36R was detected by Biacore T200, and all processes were carried out at 25°C.
  • a commercial Protein A chip was used to fix an appropriate amount of antibody by capture method, so that Rmax was around 50RU, and the capture flow rate was 10 ⁇ l/min.
  • the antigen was gradient diluted, and the instrument flow rate was switched to 30 ⁇ l/min.
  • the reference channel and the channel of the fixed antibody were flowed in order from low to high concentration, and the buffer was flowed as a negative control. After each binding and dissociation, the chip was regenerated with pH1.5 glycine.
  • the 1:1 binding model in the Kinetics option was selected in the instrument's own analysis software for fitting, and the antibody binding rate constant k a , dissociation rate constant k d and dissociation equilibrium constant K D values were calculated.
  • the data in the table are: each sample was tested three times and the average value was calculated.
  • Example 3 QX009N (HZD25-54) and Spesolimab analogs neutralize STAT3 phosphorylation activity induced by human IL-36 ( ⁇ , ⁇ , ⁇ ) in HT29 reporter cells
  • the HT29 reporter gene cell line was used to determine the activity of QX009N (HZD25-54) in antagonizing the intracellular signaling molecule STAT3 phosphorylation mediated by human IL-36 ( ⁇ , ⁇ , ⁇ ) through IL-36R-IL-1RAcp: 40,000 cells were added to a 96-well cell culture plate at a volume of 100 ⁇ l per well, and then cultured overnight at 37°C and 5% CO 2. Antibodies were added to the cells for incubation for 1 hour, where the final concentration of the antibodies ranged from 0 to 10,000 ng/ml.
  • recombinant human IL-36 mixture (containing 2 ng/ml recombinant human IL-36 ⁇ , 1 ng/ml recombinant human IL-36 ⁇ , and 40 ng/ml recombinant human IL-36 ⁇ ) was added, and then cultured for 24 hours at 37°C and 5% CO 2 .
  • the cell culture supernatant was discarded, and 120 ⁇ l of ONE-Glo-Luciferase Reagent was added to each well for 10 min. 80 ⁇ l was taken from each well to a white 96-well plate, and 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.
  • the results shown in Figure 4 show that QX009N (HZD25-54) can inhibit human IL-36 ( ⁇ , ⁇ , ⁇ )-induced STAT3 phosphorylation in HT29 reporter gene cells.
  • the IC50 of QX009N (HZD25-54) in inhibiting the STAT3 phosphorylation activity of human IL-36 ( ⁇ , ⁇ , ⁇ )-induced HT29 reporter gene cells is 2.16 ng/ml
  • the IC50 of Spesolimab analogs in inhibiting the STAT3 phosphorylation of human IL-36 ( ⁇ , ⁇ , ⁇ )-induced HT29 reporter gene cells is 15.67 ng/ml.
  • Example 4 QX009N (HZD25-54) and Spesolimab analogs neutralize the activity of CXCL-1 and IL-8 release induced by human IL-36 ( ⁇ , ⁇ , ⁇ ) in HT29 cells
  • the HT29 cell line was used to determine whether QX009N (HZD25-54) antagonized human IL-36 ( ⁇ , ⁇ , ⁇ ) and induced HT29 cells to release CXCL-1 and IL-8 through IL-36R-IL-1RAcp: 40,000 cells were added to a 96-well cell culture plate at a volume of 100 ⁇ l per well, and then cultured overnight at 37°C and 5% CO 2. Antibodies were added to the cells for incubation for 1 hour, where the final concentration of the antibodies ranged from 0 to 10,000 ng/ml.
  • a recombinant human IL-36 mixture (containing 1 ng/ml recombinant human IL-36 ⁇ , 0.2 ng/ml recombinant human IL-36 ⁇ , and 4 ng/ml recombinant human IL-36 ⁇ ) was added, and then cultured at 37°C and 5% CO 2 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 method, and the dose-effect curve was drawn to analyze the antagonistic activity of the antibody.
  • the dose-effect curve is shown in Figure 5.
  • 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 ( ⁇ , ⁇ , ⁇ ) from HT29 cells.
  • the IC50s of QX009N (HZD25-54) for inhibiting the release of CXCL-1 and IL-8 induced by human IL-36 ( ⁇ , ⁇ , ⁇ ) from HT29 cells are 2.21 ng/ml and 1.53 ng/ml, respectively.
  • the IC50s of Spesolimab analogs for inhibiting the release of CXCL-1 and IL-8 induced by human IL-36 ( ⁇ , ⁇ , ⁇ ) from HT29 cells are 12.29 ng/ml and 11.47 ng/ml, respectively.
  • Example 5 QX009N (HZD25-54) and Spesolimab analogs neutralize the activity of A431 cells releasing CXCL-1 and IL-8 induced by human IL-36 ( ⁇ , ⁇ , ⁇ )
  • A431 cell line was used to measure QX009N (HZD25-54) antagonizing human IL-36 ( ⁇ , ⁇ , ⁇ ) to induce A431 cells to release CXCL-1 and IL-8 through IL-36R-IL-1RAcp: 40,000 cells were added to a 96-well cell culture plate at a volume of 100 ⁇ l per well, and then cultured overnight at 37°C and 5% CO 2. Antibodies were added to the cells for incubation for 1 hour, where the final concentration of the antibodies ranged from 0 to 10,000 ng/ml.
  • recombinant human IL-36 mixture (containing 20 ng/ml recombinant human IL-36 ⁇ , 2 ng/ml recombinant human IL-36 ⁇ , and 50 ng/ml recombinant human IL-36 ⁇ ) was added, and then cultured at 37°C and 5% CO 2 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 method, and the dose-effect curve was drawn to analyze the antagonistic activity of the antibody.
  • the dose-effect curve is shown in Figure 6.
  • the IC 50s of Spesolimab analogs that inhibit the release of CXCL-1 and IL-8 induced by A431 cells by human IL-36 are 22.46 ng/ml and 16.02 ng/ml, respectively.
  • Example 6 QX009N (HZD25-54) and Spesolimab analogs neutralize the activity of human IL-36 ⁇ in inducing IL-8 release from human PBMC cells
  • Human PBMC cells were used to determine the activity of QX009N (HZD25-54) in antagonizing human IL-36 ⁇ to induce 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 100 ⁇ l and 200,000 cells of PBMCs were added to a 96-well cell culture plate at each well. Antibodies were added to the PBMCs and incubated for 1 hour, wherein the final concentration of the antibodies ranged from 0 to 5000 ng/ml.

Abstract

本发明提供一种抗人白介素36受体(IL-36R)的单克隆抗体及其应用。所述单克隆抗体包含三个重链互补决定区CDR-H1、CDR-H2和CDR-H3和三个轻链互补决定区CDR-L1、CDR-L2和 CDR-L3，所述CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2和 CDR-L3的氨基酸序列分别如 SEQ ID NO: 1至SEQ ID NO: 6所示。所述单克隆抗体与Spesolimab相比，与人IL-36R结合的亲和力相当，细胞水平的中和活性优于后者。

Description

抗人白介素36受体单克隆抗体及其应用 技术领域
本申请涉及抗体药物领域。具体地，本申请涉及针对人白介素36受体(IL-36R)单克隆抗体及其应用。
背景技术
白细胞介素-36(IL-36)，属于IL-1家族(IL-1family，IL1F)，由IL-36受体激动剂IL-36α(IL1F6)、IL-36β(IL1F8)、IL-36γ(IL1F9)和IL-36受体拮抗剂IL-36Ra(IL-36 receptor antagonist，IL1F5)组成，其结构模式和经典的IL-1家族相似：缺乏信号肽，无法通过经典的高尔基体-内质网途径分泌，通常先形成无活性的前体，继而被蛋白酶水解活化。IL-36可由单核细胞、巨噬细胞、T/B淋巴细胞、角质细胞、上皮细胞等多种细胞产生。IL-36的受体由特异性受体IL-36R(亦称IL-1RL2，Interleukin-1 receptor-like 2)和IL-1受体辅助蛋白IL-1RAcP(IL-1 receptor accessory protein)组成，皆由3个结构域构成的胞外区，跨膜区、胞内Toll/IL-1受体(Toll/IL-1 receptor，TIR)结构域组成。蛋白水解成熟的激动型IL-36配体(α、β、γ)与IL-36R结合形成二元复合物，进而招募IL-1RAcP，组装成功能性三元复合物，IL-36R和IL-1RAcP的胞内TIR相互作用，聚集转导接头蛋白髓样分化蛋白88(Myeloid differentiation factor 88，MyD88)以及白细胞介素-1受体相关激酶(IL-1 receptor associated kinase，IRAK)进而激活下游NF-κB(Nuclear factor kappa-B，核因子κB)、MAPK(Mitogen-activated protein kinase，丝裂原活化蛋白激酶)信号通路。当IL-36正常表达时，其可通过炎症反应进行宿主防御，当IL-36表达失调时，会刺激其受体表达细胞(上皮细胞、成纤维细胞、角质细胞、单核细胞、巨噬细胞、树突状细胞、T细胞)产生炎性细胞因子、趋化因子、黏附分子等，进而介导病理性的炎症反应，参与泛发性脓疱型银屑病、掌跖脓疱病、特应性皮炎、炎症性肠病、慢性阻塞性肺病等慢性炎症和自身免疫性疾病的病理过程。
[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.
[2]Gabay C,Towne J E.Regulation and function of interleukin-36 cytokines in homeostasis and pathological conditions[J].Journal of Leukocyte Biology,2015,97(4):645.
[3]Henry 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.
[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.
[5]Buhl,A L,Wenzel,J.Interleukin-36 in Infectious and Inflammatory Skin Diseases[J].Frontiers in Immunology,2019.01162
发明内容
本申请的目的在于提供一种新的抗人IL-36R单克隆抗体、包含该单克隆抗体的药物组合物以及该单克隆抗体的制药用途。
本申请的技术方案如下所述：
1.一种抗人IL-36R单克隆抗体，其包含三个重链互补决定区(CDR-H1、CDR-H2以及CDR-H3)和三个轻链互补决定区(CDR-L1、CDR-L2以及CDR-L3)，其中：
CDR-H1(在本说明书中CDR-H1表示重链CDR1)的氨基酸序列如SEQ ID NO：1(NYAMG)所示；
CDR-H2(在本说明书中CDR-H2表示重链CDR2)的氨基酸序列如SEQ ID NO：2(YISGGGSAYYASWAKG)所示；
CDR-H3(在本说明书中CDR-H3表示重链CDR3)的氨基酸序列如SEQ ID NO：3(WAIKSYFFGMDL)所示；
CDR-L1(在本说明书中CDR-L1表示轻链CDR1)的氨基酸序列如SEQ  ID NO：4(QASEYISSYLA)所示；
CDR-L2(在本说明书中CDR-L2表示轻链CDR2)的氨基酸序列如SEQ ID NO：5(QASTLAS)所示；
CDR-L3(在本说明书中CDR-L3表示轻链CDR3)的氨基酸序列如SEQ ID NO：6(QTNNAIHTYGGA)所示。
2.根据项1所述的单克隆抗体，其包含重链可变区和轻链可变区，其中，
所述重链可变区的氨基酸序列如SEQ ID NO：7所示，其氨基酸序列为EVQLVESGGGLVQPGGSLRLSCAASGIDLSNYAMGWVRQAPGKGLEWVGYISGGGSAYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWAIKSYFFGMDLWGQGTLVTVSS；
所述轻链可变区的氨基酸序列如SEQ ID NO：8所示，其氨基酸序列为DIQMTQSPSSVSASVGDRVTITCQASEYISSYLAWYQQKPGKAPKLLIYQASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQTNNAIHTYGGAFGGGTKVEIK。
3.一种分离的核酸，其编码前述任一单克隆抗体。
4.一种宿主细胞，其包含根据项3所述的核酸。
所述核酸可以存在于载体上。载体可以属于任意类型，例如，重组载体诸如表达载体。可以使用多种宿主细胞中的任一种。在一个实施方案中，宿主细胞是原核细胞，例如，大肠杆菌(E.coli)。在另一个实施方案中，宿主细胞是真核细胞，例如，哺乳动物细胞，诸如中国仓鼠卵巢(CHO)细胞。
5.一种生产单克隆抗体的方法，所述方法包括培养根据项4所述的宿主细胞从而生产前述任一单克隆抗体。
所述方法包括在合适的宿主细胞中表达编码所述抗人IL-36R单克隆抗体的重组载体，从而生产所述单克隆抗体。在某些实施方案中，所述方法包括培养包含编码所述抗人IL-36R单克隆抗体的核酸的宿主细胞，从而表达所述核酸。所述方法可以进一步包括从宿主细胞培养物或宿主细胞培养基回收所述抗人IL-36R单克隆抗体。
6.一种药物组合物，其包含前述任一单克隆抗体和药学上可接受的载体。
所述药物组合物可以进一步包含另外的治疗剂(例如，不同的抗人 IL-36R抗体)。
7.根据项6所述的药物组合物，其用于治疗IL-36R介导的信号转导相关疾病。
8.根据项7所述的药物组合物，其中，所述IL-36R介导的信号转导相关疾病选自皮炎、银屑病、炎性肠病、关节炎、***性红斑狼疮、炎性肺病和慢性肾病，
优选地，所述IL-36R介导的信号转导相关疾病选自泛发性脓疱型银屑病、掌跖脓疱病、特应性皮炎、炎症性肠病、慢性阻塞性肺病、寻常型银屑病、银屑病关节炎、多发性硬化症、类风湿性关节炎、硬皮病、哮喘或强直性脊柱炎。
9.前述任一单克隆抗体在制备用于治疗IL-36R介导的信号转导相关疾病的药物中的用途。
10.根据项9所述的用途，其中，所述IL-36R介导的信号转导相关疾病选自皮炎、银屑病、炎性肠病、关节炎、***性红斑狼疮、炎性肺病和慢性肾病，
优选地，所述IL-36R介导的信号转导相关疾病选自泛发性脓疱型银屑病、掌跖脓疱病、特应性皮炎、炎症性肠病、慢性阻塞性肺病、寻常型银屑病、银屑病关节炎、多发性硬化症、类风湿性关节炎、硬皮病、哮喘或强直性脊柱炎。
11.一种治疗IL-36R介导的信号转导相关的疾病的方法，其包括：
向有此需要的受试者给药根据前述任一项所述的单克隆抗体或根据前述任一项所述的药物组合物。
12.根据项11所述的方法，其中，所述IL-36R介导的信号转导相关疾病选自皮炎、银屑病、炎性肠病、关节炎、***性红斑狼疮、炎性肺病和慢性肾病，
优选地，所述IL-36R介导的信号转导相关疾病选自泛发性脓疱型银屑病、掌跖脓疱病、特应性皮炎、炎症性肠病、慢性阻塞性肺病、寻常型银屑病、银屑病关节炎、多发性硬化症、类风湿性关节炎、硬皮病、哮喘或强直性脊柱炎。
本申请提供了一种新的抗人IL-36R单克隆抗体，其与现有技术中的抗 人IL-36R单克隆抗体Spesolimab相比，结合IL-36R的亲和力相当，且细胞水平的中和活性优于Spesolimab。需要说明的是，Spesolimab为勃林格殷格翰公司研发的靶向IL-36R的单克隆抗体药物，Spesolimab治疗成人泛发性脓疱型银屑病的三期临床NAVIGATOR获得成功，目前已在美国食品药品监督管理局获得上市批准，并已在中国国家药品监督管理局获得优先评审资格。本申请的单克隆抗体(例如QX009N(HZD25-54))可以为IgG1(免疫球蛋白G1)型靶向IL-36R(白介素-36受体)的人源化单克隆抗体，对IL-36R具有高亲和力，能特异性结合IL-36R，阻断IL-36炎症通路信号。HZD25-54与IL-36R结合，竞争性阻断受体激动剂(IL-36α、β、γ)与IL-36R的结合，下调下游促炎信号通路和促纤维化信号通路，抑制上皮细胞/成纤维细胞/免疫细胞介导的炎症反应，从而减少炎性疾病/皮肤疾病中驱动致病的细胞炎症因子的释放，达到控制疾病的目的。
本申请的单克隆抗体，在细胞水平显示出优于Spesolimab(根据专利公开序列表达制备)的中和活性，其有望在预防和治疗相关疾病方面展现出良好的临床效果。
附图说明
附图用于更好地理解本申请，不构成对本申请的不当限定。其中：
图1是显示构建HZD25-54瞬转表达质粒的核酸电泳结果的图。其中，M：Marker；条带1：PCR产物25VH-Hu12；条带2：pQX1，HindIII/BamHI；条带3：PCR产物25VK-Hu17；条带4：pQX2.3，HindIII/BsiWI。
图2是瞬转表达流程图。
图3是QX009N(HZD25-54)的电泳检测图。
图4是显示QX009N(HZD25-54)和Spesolimab类似物中和人IL-36(α、β、γ)诱导HT29报告基因细胞中STAT3磷酸化活性的图。
图5是显示QX009N(HZD25-54)和Spesolimab类似物中和人IL-36(α、β、γ)诱导的HT29细胞释放CXCL-1、IL-8活性的图。
图6是显示QX009N(HZD25-54)和Spesolimab类似物中和人IL-36(α、β、γ)诱导的A431细胞释放CXCL-1、IL-8活性的图。
图7是显示QX009N(HZD25-54)和Spesolimab类似物中和人IL-36β诱导PBMC细胞释放IL-8活性的图。
具体实施方式
以下对本申请的示范性实施例做出说明，其中包括本申请实施例的各种细节以助于理解，应当将它们认为仅仅是示范性的。因此，本领域普通技术人员应当认识到，可以对这里描述的实施例做出各种改变和修改，而不会背离本申请的范围和精神。同样，为了清楚和简明，以下的描述中省略了对公知功能和结构的描述。
本说明书中提及的科技术语具有与本领域技术人员通常理解的含义相同的含义，如有冲突以本说明书中的定义为准。
一般而言，本说明书中采用的术语具有如下含义。
在本说明书中，“分离的”抗体是已经与它的天然环境的组分分离的抗体。在某些实施方案中，将抗体纯化至大于95％或99％纯度，所述纯度通过例如电泳(例如，SDS-PAGE等电聚焦(IEF)、毛细管电泳)或色谱(例如，离子交换或反相HPLC)来确定。关于评价抗体纯度的方法的综述，参见例如Flatman等，J.Chromatogr.B848：79-87(2007)。
在本说明书中，“单克隆抗体”表示得自基本上同源的抗体的群体的抗体，即，构成所述群体的各个抗体是相同的和/或结合相同表位，除了可能的变体抗体(例如，含有天然存在的突变或在单克隆抗体制品的生产过程中产生)以外，这样的变体通常以微量存在。与通常包括针对不同决定簇(表位)的不同抗体的多克隆抗体制品不同，单克隆抗体制品的每种单克隆抗体针对抗原上的单个决定簇。因而，修饰语“单克隆”指示所述抗体得自基本上同源的抗体群体的特征，并且不应解释为需要通过任何特定方法生产所述抗体。例如，要根据本申请使用的单克隆抗体可以通过多种技术来制备，所述技术包括、但不限于杂交瘤方法、重组DNA方法、噬菌体展示方法、和使用包含人免疫球蛋白基因座的全部或部分的转基因动物的方法，本文描述了这样的方法和其它示例性的制备单克隆抗体的方法。
在本说明书中，“亲和力”表示分子(例如，抗体)的单个结合位点和它的结合配偶体(例如，抗原)之间的非共价相互作用的总和的强度。除非另外指出，否则本说明书中使用的“结合亲和力”表示反映结合对(例如，抗体和抗原)的成员之间的1∶1相互作用的固有结合亲和力。分子X对它的配偶体Y的亲和力通常可以由平衡解离常数(K D)表示。通过本领域已知的常见方法， 可以测量亲和力。
在本说明书中，人白介素-36受体(Human Interleukin-36 Receptor，hIL-36R，有些情况下也简写作IL-36R)表示一种源自人的膜受体，其胞外区氨基酸序列如SEQ ID NO：9所示，其中，下划线部分表示信号肽。
SEQ ID NO：9：
Figure PCTCN2022134732-appb-000001
在本说明书中，“抗人IL-36R单克隆抗体”表示这样的单克隆抗体：其能够以足够的亲和力结合人IL-36R，使得所述单克隆抗体可用作靶向人IL-36R的诊断剂和/或治疗剂。
本申请的抗人IL-36R单克隆抗体与靶标无关的蛋白不结合。这里，“无关的蛋白”是指除作为靶标的人IL-36R以外的其他蛋白；这里，“不结合”是指：在将本申请的抗人IL-36R单克隆抗体与作为其靶标的人IL-36R的结合能力作为100％的情况下，本申请的抗人IL-36R单克隆抗体与所述无关蛋白的结合能力小于10％，例如9％、8％、7％、6％、5％、4％、3％、2％、1％或者0。
本申请的抗人IL-36R单克隆抗体与其他动物种属的IL-36R可以不结合。这里，“其他动物种属”是指除人以外的其他动物种属，例如狨猴、食蟹猴、猪、犬、兔、大鼠、小鼠、豚鼠等；这里，“不结合”是指：在将本申请的抗人IL-36R单克隆抗体与作为其靶标的人IL-36R的结合能力作为100％的情况下，本申请的抗人IL-36R单克隆抗体与其他动物种属的IL-36R的结合能力小于10％，例如9％、8％、7％、6％、5％、4％、3％、2％、1％或者0。
本申请的人IL-36R单克隆抗体可以具有例如≤1μM、≤100nM、≤50nM、≤40nM的平衡解离常数(K D)。
实验结果显示，本申请的抗人IL-36R单克隆抗体可以特异性结合人 IL-36R。
本申请的抗人IL-36R单克隆抗体在诸多生物活性方面与上市同类单抗产品相当、或优于上市同类单抗产品。所述生物活性例如中和人IL-36(α、β、γ)诱导细胞中STAT3磷酸化的活性、中和人IL-36(α、β、γ)诱导细胞释放CXCL-1、IL-8的活性、中和人IL-36β诱导人PBMC细胞释放IL-8的活性等。
在一个具体实施方式中，本申请的抗人IL-36R单克隆抗体的重链的氨基酸序列可以如SEQ ID NO：10所示；轻链的氨基酸序列可以如SEQ ID NO：11所示。
SEQ ID NO：10
Figure PCTCN2022134732-appb-000002
SEQ ID NO：11
Figure PCTCN2022134732-appb-000003
其中，SEQ ID NO：10和11均为经人源化的序列。为了去除抗体的ADCC和CDC效应，重链恒定区进行了LALA突变。
在本说明书中，“分离的”核酸表示已经与它的天然环境的组分分离的核酸分子。分离的核酸包括通常含有核酸分子的细胞中包含的核酸分子，但是所述核酸分子存在于染色体外或在不同于其天然染色***置的染色*** 置。
在本说明书中，“分离的编码抗IL-36R单克隆抗体的核酸”表示编码抗体重链和轻链的一个或多个核酸分子，包括在单个载体或分开的载体中的这样的核酸分子、以及存在于宿主细胞中的一个或多个位置的这样的核酸分子。
在本说明书中，“载体”表示能够扩增与其连接的另一核酸的核酸分子。该术语包括作为自我复制的核酸结构的载体以及整合进它已经引入其中的宿主细胞的基因组中的载体。某些载体能够指导与其可操作地连接的核酸的表达。这样的载体在本文被称为“表达载体”。
在本说明书中，“宿主细胞”、“宿主细胞系”和“宿主细胞培养”可互换使用，且表示其中已经引入外源核酸的细胞，包括这种细胞的后代。宿主细胞包括“转化体”和“转化的细胞”，其包括原代转化的细胞和由其来源的后代(不考虑传代数)。后代在核酸内容物方面可以与亲本细胞不完全相同，但是可以含有突变。针对最初转化的细胞筛选或选择的具有相同功能或生物活性的突变体后代被包括在本说明书中。
在本说明书中，“药物组合物”表示这样的制品：其呈现使得包含在其中的活性成分的生物活性能够发挥效果的形式，并且所述组合物不含有对所述制剂要施用的受试者有不可接受的毒性的额外组分。
在本说明书中，“药学上可接受的载体”表示药物组合物中除了活性成分之外的成分，其对受试者无毒。药学上可接受的载体包括、但不限于缓冲剂、赋形剂、稳定剂或防腐剂。
在本申请书中，“单克隆抗体”一般为人抗体，其可以使用本领域技术人员公知的技术来制备，例如，人抗体一般描述于van Dijk,M.A.and van de Winkel,J.G.,Curr.Opin.Pharmacol.5:368-374(2001)及Lonberg,N.,Curr.Opin.Immunol.20:450-459(2008)。
可以通过向已经经过修饰而对抗原攻击刺激生产完整人抗体或具有人类可变区的完整抗体的转基因动物施用免疫原来制备抗体，这些动物通常含有一部分或全部的人类免疫球蛋白基因座，其替换了内源免疫球蛋白基因座，或者存在于染色体外或随机整合于动物体内。在此类转基因小鼠中，内源免疫球蛋白基因座一般已经失活，关于自转基因动物获得人抗体的方法的综述，参见Lonberg,N.,Nat.Biotech.(自然生物技术) 23:1117-1125(2005)。还可参见例如美国专利No.6,075,181和No.6,150,584描述的XENOMOUSE TM技术；美国专利No.5,770,429描述的
Figure PCTCN2022134732-appb-000004
技术；美国专利No.7,041,870描述的
Figure PCTCN2022134732-appb-000005
技术，和美国专利申请公开文本No.US 2007/0061900描述的
Figure PCTCN2022134732-appb-000006
技术。可以例如通过与不同人恒定区组合进一步修饰来自由此类动物生成的完整抗体的人可变区。
还可以通过基于杂交瘤的方法来制备人抗体。已描述了用于生产人单克隆抗体的人骨髓瘤和小鼠-人杂交骨髓瘤细胞(参见例如Kozbor,D.,J.Immunol.133:3001-3005(1984)；Brodeur,B.R.等，Monoclonal Antibody Production Techniques and Applications,Marcel Dekker,Inc.,New York(1987),pp.51-63；Boerner,P.等，J.Immunol.147:86-95(1991))。经由人B细胞杂交瘤技术生产的人抗体也记载于Li,J.等，Proc.Natl.Acad.Sci.USA103:3557-3562(2006)。其他方法包括那些记载于例如美国专利No.7,189,826(其描述了自杂交瘤细胞系生成单克隆人IgM抗体)以及Ni,Xiandai Mianyixue,26(4)；265-268(其描述了人-人杂交瘤)的。人杂交瘤技术(Trioma技术)也记载于Vollmers,H.P.and Brandlein,S.,Histology and Histopathology 20:927-937(2005)；Vollmers,H.P.and Brandlein,S.,Methods and Findings in Experimentaland Clinical Pharmacology 27:185-191(2005)。
还可通过分离选自来源于人的噬菌体展示文库的Fv克隆可变结构域序列来生成人抗体，然后，可以将此类可变域序列与期望的人恒定域组合。
还可以基于自抗体文库选择人抗体，即可以通过对组合文库筛选具有期望的一种或多种活性的抗体来分离人抗体。例如，用于生产噬菌体展示文库及对此类文库筛选拥有期望结合特征的抗体的多种方法是本领域已知的。这种方法综述于例如Hoogenboom,H.R.等，Methods in Molecular Biology 178:1-37(2001)，并且进一步记载于例如McCafferty,J.等，Nature 348:552-554(1990)；Clackson,T.等，Nature 352:624-628(1991)；Marks,J.D.等，J.Mol.Biol.222:581-597(1992)；Marks,J.D.and Bradbury,A.,Methods in Molecular Biology 248:161-175(2003)；Sidhu,S.S.等，J.Mol.Biol.338:299-310(2004)；Lee,C.V.等，J.Mol.Biol.340:1073-1093(2004)；Fellouse,F.A.,Proc.Natl.Acad.Sci.USA 101:12467-12472(2004)；及Lee,C.V.等，J.Immunol.Methods 284:119-132(2004)。
在某些噬菌体展示方法中，通过聚合酶链反应(PCR)分别克隆VH和VL基因的全集，并在噬菌体文库中随机重组，然后在所述噬菌体文库中筛选抗原结合性噬菌体，如记载于Winter,G.等，Ann.Rev.Immunol.12:433-455(1994)。噬菌体通常以单链Fv(scFv)片段或以Fab片段展示抗体片段。来自经免疫来源的文库提供针对免疫原的高亲和力抗体，而不需要构建杂交瘤。或者，可以(例如自人)克隆未免疫全集以在没有任何免疫的情况中提供针对一大批非自身和还有自身抗原的抗体的单一来源，如由Griffiths,A.D.等，EMBO J,12:725-734(1993)描述的。最后，也可以通过从干细胞克隆未重排的V基因区段，并使用含有随机序列的PCR引物编码高度可变的CDR3区并在体外实现重排来合成生成未免疫文库，如由Hoogenboom,H.R.and Winter,G.,J.Mol.Biol.227:381-388(1992)所描述的。描述人抗体噬菌体文库的专利公开文本包括例如：美国专利No.5,750,373及美国专利公开文本No.2005/0079574、2005/0119455、2005/0266000、2007/0117126、2007/0160598、2007/0237764、2007/0292936和2009/0002360。
所述抗体也可以是多特异性抗体，例如双特异性抗体。双特异性抗体是对至少两种不同位点具有结合特异性的单克隆抗体。用于生成多特异性抗体的技术包括但不限于具有不同特异性的两对免疫球蛋白重链-轻链的重组共表达(参见Milstein,C.and Cuello,A.C.,Nature305:537-540(1983)；WO 93/08829；及Traunecker,A.等，EMBO J.10:3655-3659(1991))和“节-入-穴”工程化(参见例如美国专利No.5,731,168)。也可以通过用于生成抗体Fc-异二聚体分子的工程化静电操纵效应(WO 2009/089004)；交联两种或更多种抗体或片段(参见例如美国专利No.4,676,980及Brennan,M.等，Science 229:81-83(1985))；使用亮氨酸拉链来生成双特异性抗体(参见例如Kostelny,S.A.等，J.Immunol.148:1547-1553(1992))；使用用于生成双特异性抗体片段的“双抗体”技术(参见例如Holliger,P.等，Proc.Natl.Acad.Sci.USA 90:6444-6448(1993))；及使用单链Fv(scFv)二聚体(参见例如Gruber,M.等，J.Immunol.152:5368-5374(1994))；及制备三特异性抗体(如例如Tutt,A.等，J.Immunol.147:60-69(1991)中所描述的)来生成多特异性抗体。
本文中所述的单克隆抗体还包括具有三个或更多个功能性抗原结合位点的工程化改造抗体，包括“章鱼抗体”(参见例如US 2006/0025576)。
本文中的抗体还可以包括WO 2009/080251、WO 2009/080252、 WO2009/080253、WO 2009/080254、WO 2010/112193、WO 2010/115589、WO2010/136172、WO 2010/145792、及WO 2010/145793、WO 2011/117330、WO 2012/025525、WO 2012/025530、WO 2013/026835、WO2013/026831、WO 2013/164325、或WO 2013/174873中记载的多特异性抗体。
本文中所述的单克隆抗体也可以是抗体变体，例如，可能期望改善抗体的结合亲和力和/或其它生物学特性。可以通过将适宜的修饰引入编码抗体的核苷酸序列中，或者通过肽合成来制备抗体的氨基酸序列变体。此类修饰包括例如对抗体的氨基酸序列内的残基的删除、和/或***和/或替代。可以进行删除、***、和替代的任何组合以得到最终的构建体，只要最终的构建体拥有期望的特征，例如抗原结合。因此，在某些实施方案中，提供了具有一个或多个氨基酸置换的抗体变体，用于置换突变的感兴趣的位点包括HVR和FR，例如，可将氨基酸置换引入感兴趣的抗体中并筛选具有所需活性的产物，例如，保留/改善的抗原结合性，降低的免疫原性，或改善的ADCC或CDC。
实施例
下述实施例中所使用的实验方法如无特殊要求，均为常规方法。
下述实施例中所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1抗人IL-36R单克隆抗体QX009N的制备
荃信生物自制表达的人白介素36受体(hIL-36R-Rabbit Fc)，作为免疫原免疫新西兰兔，运用B细胞克隆技术获得抗原结合特异性抗体克隆，进而筛选出结合人IL-36R并具有人IL-36R抑制活性的单克隆抗体。通过Binding ELISA以及HT29细胞释放IL-8的方法检测细胞上清进行分析、筛选，挑选目标克隆。以上免疫和筛选过程委托给商业化公司完成。
先后挑选出10个克隆进行重组表达，并测序。经测定，25#克隆的细胞中和活性最好。因此，对25#克隆进行人源化改造。利用NCBI IgBlast进行人IgG胚系序列(Germline)同源性比对，选择IGHV3-66*01作为重链CDR移植模板，将25#克隆重链的CDR区(即CDR-H1(SEQ ID No:1)、CDR-H2(SEQ ID No:2)和CDR-H3(SEQ ID No:3))移植入IGHV3-66*01的骨 架区；选择IGKV1-12*01作为轻链CDR移植模板，将25#克隆轻链的CDR区(即CDR-L1(SEQ ID No:4)、CDR-L2(SEQ ID No:5)和CDR-L3(SEQ ID No:6))移植入IGKV1-12*01的骨架区；对骨架区特定位点进行回复突变和CDR区定点突变，获得本申请的单克隆抗体QX009N可变区。最终，人源化后的重链可变区序列如SEQ ID NO：7所示；人源化后的轻链可变区氨基酸序列如SEQ ID NO：8所示。
上述重链(SEQ ID NO：10)的基因和轻链可变区(SEQ ID NO：8)的基因，利用PCR扩增获得。用HindIII和BamHI双酶切重链表达质粒pQX1；用HindIII和BsiWI双酶切轻链表达质粒pQX2.3；用Infusion重组酶将PCR扩增基因分别***对应的表达质粒中，构建重链表达质粒pQX2.1-25VH-Hu12和轻链表达质粒pQX2.3-25VK-Hu17。
通过核酸电泳检测PCR扩增的重链基因片段、轻链可变区基因片段和双酶切的质粒结果如图1所示。根据图1的结果可以看出，抗体重链、轻链可变区PCR扩增结果以及双酶切重链和轻链表达质粒的结果，其中，重链和轻链的质粒大小约5000bp，重链约1469bp，轻链可变区约441bp。
将序列正确的重链表达质粒pQX2.1-25VH-Hu12(所表达的重链的氨基酸序列如SEQ ID NO：10所示)和轻链表达质粒pQX2.3-25VK-Hu17(所表达的轻链的氨基酸序列如SEQ ID NO：11所示)共转染ExpiCHO-S细胞。转染前一天，将ExpiCHO-S细胞稀释成3×10 6个细胞/ml进行转染前传代。转染当天，将细胞密度稀释成6×10 6个细胞/ml，125ml摇瓶装25ml细胞，等待转染。转染和表达过程如图2所示。
转染后第6天，收获培养上清，用ProteinA进行一步纯化。用SDS-PAGE电泳检测纯化的抗体，将其命名为QX009N(HZD25-54)，利用蛋白电泳检测该抗体的结果如图3所示。蛋白电泳用变性还原胶检测，图3的结果显示出有两条带，两个条带的大小分别约50kDa和25kDa，与重链(49.0kDa)和轻链(23.4kDa)理论分子量一致。
实施例2平衡解离常数(K D)的测定
用Biacore T200检测QX009N(HZD25-54)与人IL-36R的亲和力，所有过程都在25℃进行。采用商品化Protein A芯片，通过捕获法固定适量的抗体，使得Rmax在50RU左右，捕获流速是10μl/min。将抗原进行梯度稀释， 仪器流速切换成30μl/min，按照浓度从低到高的顺序依次流过参比通道和固定抗体的通道，流过缓冲液作为阴性对照。每一个结合、解离完成后用pH1.5甘氨酸再生芯片。用仪器自带分析软件选择Kinetics选项中1:1结合模型进行拟合，计算抗体的结合速率常数k a，解离速率常数k d以及解离平衡常数K D值。
除此之外，将QX009N(HZD25-54)与目前已经处于临床III期的针对人IL-36R的单克隆抗体，即Spesolimab的亲和力进行比较，针对已知抗体的检测方法与对QX009N进行检测的方法相同，结果如表1所示。其中Spesolimab根据专利US9023995B2提供的B6序列，构建表达质粒，瞬转ExpiCHO-S细胞自制获得。
表1抗人IL-36R单克隆抗体结合人IL-36R的亲和力
样品名称 k a(10 5M -1S -1) k d(10 -5S -1) K D(10 -10M)
QX009N 1.14 3.58 3.13
Spesolimab 0.36 3.11 8.62
表中的数据为：每个样品检测三次，计算平均值的数据。
实施例3 QX009N(HZD25-54)和Spesolimab类似物中和人IL-36(α、β、γ)诱导的HT29报告基因细胞STAT3磷酸化活性
利用HT29报告基因细胞系测定QX009N(HZD25-54)拮抗人IL-36(α、β、γ)通过IL-36R-IL-1RAcp介导的胞内信号分子STAT3磷酸化活性：以每孔100μl体积、40000个细胞加入到96孔细胞培养板内，然后在37℃和5％CO 2条件下培养过夜。向细胞中加入抗体孵育1h，其中抗体的终浓度范围为0至10000ng/ml，孵育完成后加入50μl/孔重组人IL-36混合物(含2ng/ml重组人IL-36α、1ng/ml重组人IL-36β、40ng/ml重组人IL-36γ)，然后在37℃和5％CO 2条件下培养24小时。弃除细胞培养上清液，每孔加入120μl ONE-Glo-Luciferase Reagent检测试剂，作用10min，每孔取80μl至白色96孔板，检测Luminescence荧光信号值并绘制剂量效应曲线，进而分析抗体的拮抗活性，剂量效应曲线如图4所示。
图4所示的结果显示，QX009N(HZD25-54)能够抑制人IL-36(α、β、γ) 诱导HT29报告基因细胞STAT3磷酸化，QX009N(HZD25-54)抑制人IL-36(α、β、γ)诱导的HT29报告基因细胞STAT3磷酸化活性的IC 50为2.16ng/ml，Spesolimab类似物抑制人IL-36(α、β、γ)诱导的HT29报告基因细胞STAT3磷酸化的IC 50为15.67ng/ml。
实施例4 QX009N(HZD25-54)和Spesolimab类似物中和人IL-36(α、β、γ)诱导的HT29细胞释放CXCL-1、IL-8的活性
利用HT29细胞系测定QX009N(HZD25-54)拮抗人IL-36(α、β、γ)通过IL-36R-IL-1RAcp诱导HT29细胞释放CXCL-1、IL-8：以每孔100μl体积、40000个细胞加入到96孔细胞培养板内，然后在37℃和5％CO 2条件下培养过夜。向细胞中加入抗体孵育1h，其中抗体的终浓度范围为0至10000ng/ml，孵育完成后加入50μl/孔重组人IL-36混合物(含1ng/ml重组人IL-36α、0.2ng/ml重组人IL-36β、4ng/ml重组人IL-36γ)，然后在37℃和5％CO 2条件下培养24小时。收集细胞培养上清，采用夹心ELISA法检测上清中CXCL-1、IL-8的表达及绘制剂量效应曲线，进而分析抗体的拮抗活性，其剂量效应曲线如图5所示。
图5所示的结果显示，QX009N(HZD25-54)能够抑制人IL-36(α、β、γ)诱导HT29细胞释放CXCL-1、IL-8，QX009N(HZD25-54)抑制人IL-36(α、β、γ)诱导HT29细胞释放CXCL-1、IL-8的IC 50分别为2.21ng/ml、1.53ng/ml，Spesolimab类似物抑制人IL-36(α、β、γ)诱导HT29细胞释放CXCL-1、IL-8的IC 50分别为12.29ng/ml、11.47ng/ml。
实施例5 QX009N(HZD25-54)和Spesolimab类似物中和人IL-36(α、β、γ)诱导的A431细胞释放CXCL-1、IL-8的活性
利用A431细胞系测QX009N(HZD25-54)拮抗人IL-36(α、β、γ)通过IL-36R-IL-1RAcp诱导A431细胞释放CXCL-1、IL-8：以每孔100μl体积、40000个细胞加入到96孔细胞培养板内，然后在37℃和5％CO 2条件下培养过夜。向细胞中加入抗体孵育1h，其中抗体的终浓度范围为0至10000ng/ml，孵育完成后加入50μl/孔重组人IL-36混合物(含20ng/ml重组人IL-36α、2ng/ml重组人IL-36β、50ng/ml重组人IL-36γ)，然后在37℃和5％CO 2条件下培养24小时。收集细胞培养上清，采用夹心ELISA法检测 上清中CXCL-1、IL-8的表达及绘制剂量效应曲线，进而分析抗体的拮抗活性，其剂量效应曲线如图6所示。
图6所示的结果显示，QX009N(HZD25-54)能够抑制人IL-36(α、β、γ)诱导A431细胞释放CXCL-1、IL-8，QX009N(HZD25-54)能够抑制人IL-36(α、β、γ)诱导A431细胞释放CXCL-1、IL-8的IC 50分别为4.12ng/ml、2.89ng/ml，Spesolimab类似物抑制人IL-36(α、β、γ)诱导A431细胞释放CXCL-1、IL-8的IC 50分别为22.46ng/ml、16.02ng/ml。
实施例6 QX009N(HZD25-54)和Spesolimab类似物中和人IL-36β诱导人PBMC细胞释放IL-8的活性
利用人PBMC细胞测定QX009N(HZD25-54)拮抗人IL-36β通过IL-36R-IL-1RAcp诱导人PBMC细胞释放IL-8活性：按密度梯度离心法从人静脉血(来源于成年健康志愿者)中分离出PBMC，将PBMC以每孔100μl、200000个细胞加入到96孔细胞培养板内，向PBMC中加入抗体孵育1h，其中抗体的终浓度范围为0至5000ng/ml，孵育完成后加入50μl/孔10ng/ml重组人IL-36β，然后在37℃和5％CO 2条件下培养24小时，收集细胞培养上清，采用夹心ELISA法检测上清中IL-8的表达及绘制剂量效应曲线，进而分析抗体的拮抗活性，其剂量效应曲线如图7所示。
从图7所示的结果显示，QX009N(HZD25-54)能够抑制人IL-36β诱导的人PBMC细胞释放IL-8，QX009N(HZD25-54)抑制人IL-36β诱导的人PBMC细胞释放IL-8活性的IC 50为4.69ng/ml，Spesolimab类似物抑制人IL-36β诱导的人PBMC释放IL-8活性的IC 50为26.59ng/ml。

Claims (10)

  1. 一种抗人白介素36受体(IL-36R)单克隆抗体，其特征在于，包含三个重链互补决定区CDR-H1、CDR-H2以及CDR-H3和三个轻链互补决定区CDR-L1、CDR-L2以及CDR-L3，其中：
    CDR-H1的氨基酸序列如SEQ ID NO：1所示；
    CDR-H2的氨基酸序列如SEQ ID NO：2所示；
    CDR-H3的氨基酸序列如SEQ ID NO：3所示；
    CDR-L1的氨基酸序列如SEQ ID NO：4所示；
    CDR-L2的氨基酸序列如SEQ ID NO：5所示；
    CDR-L3的氨基酸序列如SEQ ID NO：6所示。
  2. 根据权利要求1所述的单克隆抗体，其特征在于，包含重链可变区和轻链可变区，其中，
    所述重链可变区的氨基酸序列如SEQ ID NO：7所示；
    所述轻链可变区的氨基酸序列如SEQ ID NO：8所示。
  3. 一种分离的核酸，其特征在于，编码根据权利要求1或2所述的单克隆抗体。
  4. 一种宿主细胞，其特征在于，包含根据权利要求3所述的核酸。
  5. 一种生产单克隆抗体的方法，其特征在于，所述方法包括培养根据权利要求4所述的宿主细胞从而生产根据权利要求1或2所述的单克隆抗体。
  6. 一种药物组合物，其特征在于，包含根据权利要求1或2所述的单克隆抗体和药学上可接受的载体。
  7. 根据权利要求6所述的药物组合物，其特征在于，用于治疗IL-36R介导的信号转导相关疾病。
  8. 根据权利要求7所述的药物组合物，其特征在于，所述IL-36R介导的信号转导相关疾病选自皮炎、银屑病、炎性肠病、关节炎、***性红斑狼疮、炎性肺病和慢性肾病，
    优选地，所述IL-36R介导的信号转导相关疾病选自泛发性脓疱型银屑病、掌跖脓疱病、特应性皮炎、炎症性肠病、慢性阻塞性肺病、寻常型银屑病、银屑病关节炎、多发性硬化症、类风湿性关节炎、硬皮病、哮喘或 强直性脊柱炎。
  9. 根据权利要求1或2所述的单克隆抗体在制备用于治疗IL-36R介导的信号转导相关疾病的药物中的用途。
  10. 根据权利要求9所述的用途，其特征在于，所述IL-36R介导的信号转导相关疾病选自皮炎、银屑病、炎性肠病、关节炎、***性红斑狼疮、炎性肺病和慢性肾病，
    优选地，所述IL-36R介导的信号转导相关疾病选自泛发性脓疱型银屑病、掌跖脓疱病、特应性皮炎、炎症性肠病、慢性阻塞性肺病、寻常型银屑病、银屑病关节炎、多发性硬化症、类风湿性关节炎、硬皮病、哮喘和强直性脊柱炎。
PCT/CN2022/134732 2022-10-20 2022-11-28 抗人白介素36受体单克隆抗体及其应用 WO2024082383A1 (zh)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104080808A (zh) * 2011-11-16 2014-10-01 勃林格殷格翰国际有限公司 抗il-36r抗体
CN107847590A (zh) * 2015-04-15 2018-03-27 安奈普泰斯生物有限公司 针对白细胞介素36受体（il‑36r）的抗体
CN112094349A (zh) * 2020-11-04 2020-12-18 上海华奥泰生物药业股份有限公司 靶向于白介素36r的抗体及其制备方法和应用
CN112513091A (zh) * 2018-07-16 2021-03-16 瑞泽恩制药公司 抗il36r抗体
CN114907477A (zh) * 2021-02-08 2022-08-16 上海普铭生物科技有限公司 抗人il-36r抗体及其应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104080808A (zh) * 2011-11-16 2014-10-01 勃林格殷格翰国际有限公司 抗il-36r抗体
CN107847590A (zh) * 2015-04-15 2018-03-27 安奈普泰斯生物有限公司 针对白细胞介素36受体（il‑36r）的抗体
CN112513091A (zh) * 2018-07-16 2021-03-16 瑞泽恩制药公司 抗il36r抗体
CN112094349A (zh) * 2020-11-04 2020-12-18 上海华奥泰生物药业股份有限公司 靶向于白介素36r的抗体及其制备方法和应用
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