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WO2022095926A1 - 靶向于白介素36r的抗体及其制备方法和应用 - Google Patents

靶向于白介素36r的抗体及其制备方法和应用 Download PDF

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Publication number
WO2022095926A1
WO2022095926A1 PCT/CN2021/128704 CN2021128704W WO2022095926A1 WO 2022095926 A1 WO2022095926 A1 WO 2022095926A1 CN 2021128704 W CN2021128704 W CN 2021128704W WO 2022095926 A1 WO2022095926 A1 WO 2022095926A1
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antibody
seq
present application
variable region
antibodies
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PCT/CN2021/128704
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English (en)
French (fr)
Inventor
冯玉杰
于海佳
韦小越
彭国媛
陈时
瞿丽丽
Original Assignee
上海华奥泰生物药业股份有限公司
华博生物医药技术（上海）有限公司
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Application filed by 上海华奥泰生物药业股份有限公司, 华博生物医药技术（上海）有限公司 filed Critical 上海华奥泰生物药业股份有限公司
Priority to US18/034,985 priority Critical patent/US20230399409A1/en
Priority to JP2023526875A priority patent/JP2023548535A/ja
Priority to EP21888618.2A priority patent/EP4242230A4/en
Priority to KR1020237017515A priority patent/KR20230098606A/ko
Priority to AU2021374696A priority patent/AU2021374696A1/en
Publication of WO2022095926A1 publication Critical patent/WO2022095926A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
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    • C12N2510/00—Genetically modified cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/715—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons
    • G01N2333/7155—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]

Definitions

  • the present application relates to the field of biomedicine, in particular to an antibody targeting interleukin 36R and a preparation method and application thereof.
  • IL-1 receptor family members contain at least 11 members, including IL1RI (IL1R1), IL1RII (IL1R2), IL1RAcP (IL1R3), ST2 (T1/IL1R4), IL18Ra (IL1Rrp/IL1R5), IL1Rrp2 (IL1RL2) /IL1R6/IL-36R), IL18Rb (AcPL/IL1R7), IL1RAPL1 (TIGIRR2/IL1R8), and TIGIRR1 (IL1R9).
  • IL-36R recognizes 3 different activating ligand cytokines, IL36 ⁇ , IL36 ⁇ , IL36 ⁇ (also known as IL-1F6, IL-1F8, IL-1F9), which trigger the expression of inflammatory cytokines. And there are two natural antagonists IL-36Ra (IL-1F5) and IL-38, they can inhibit the expression of inflammatory cytokines when combined with IL-36R.
  • IL-36R is expressed on lung epithelial cells, cerebrovascular cells, kidney, testis, monocytes, skin-derived keratinocytes, fibroblasts and endothelial cells.
  • IL-36R is composed of extracellular ligand binding domain, transmembrane helix and intracellular signal transduction Toll/IL-1 receptor domain (Toll/IL-1 receptor domain, TIR domain).
  • IL-36 ⁇ , IL-36 ⁇ , and IL-36 ⁇ first combine with IL-36R to form a signal transduction complex with IL-1RAcP, and then recruit myeloid differentiation factor 88 (MyD88), which activates c -C-Jun N-terminal kinases (JNK) and extracellular regulated protein kinases (ERK1/2) mediated mitogen-activated protein kinases (MAPK) and nuclear factor kappa B (NF- ⁇ B) pathway, produce a large number of inflammatory mediators, and then mediate inflammatory response, and play an important role in adaptive immunity.
  • MyD88 myeloid differentiation factor 88
  • JNK c -C-Jun N-terminal kinases
  • ERK1/2 extracellular regulated protein kinases
  • MAPK mitogen-activated protein kinases
  • NF- ⁇ B nuclear factor kappa B pathway
  • IL-36Ra The binding of IL-36Ra to IL-36R does not lead to the recruitment of IL-1RAcP, the pro-inflammatory cascade is not turned on, and the anti-inflammatory properties of IL-36Ra are realized.
  • IL-36 ⁇ , IL-36 ⁇ , IL-36 ⁇ -mediated signal activation and IL-36Ra-mediated signal inhibition are in a balanced state.
  • IL-36Ra When IL-36Ra is mutated, it will lead to its inhibitory function. Inactivation, a state of equilibrium is disrupted, and inflammatory diseases occur.
  • GPP generalized pustular psoriasis
  • PPP palmoplantar pustulosis
  • GPP is a severe systemic type of pustular psoriasis that is lethal.
  • PPP is a chronic pustular psoriasis that affects the palms and soles of the feet.
  • Current treatments for GPP and PPP include oral retinoids and topical steroids, but these treatments have poor efficacy and serious side effects.
  • anti-IL-36R antibodies that can bind to IL-36R with high affinity and that can block the binding of IL-36 ⁇ , IL-36 ⁇ , IL-36 ⁇ to IL-36R, for the treatment of ILs such as GPP and PPP -36 related diseases.
  • the purpose of the present application is to provide an antibody targeting interleukin 36R and its preparation method and application.
  • the purpose of the present application is to provide a high-affinity and high-biologically active IL-36R antibody and its application, which can bind to IL-36R with high affinity, and can block IL-36 ⁇ , IL-36 ⁇ , IL-36 ⁇ and IL-36 ⁇ . IL-36R binding.
  • Another object of the present application is to provide an interleukin 36R binding molecule and use thereof, especially in the treatment and/or prevention, or diagnosis of IL-36-related diseases such as psoriasis.
  • a first aspect of the present application provides a heavy chain variable region of an antibody, the heavy chain variable region comprising the following three complementarity determining region CDRs:
  • any one of the above amino acid sequences also includes at least one (such as 1-3, can be 1-2, can be 1) that is optionally added, deleted, modified and/or substituted. ) amino acids and derived sequences capable of retaining IL-36R binding affinity.
  • the heavy chain variable region further comprises a human FR region or a murine FR region.
  • the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:1.
  • the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:9.
  • a second aspect of the present application provides a heavy chain of an antibody, wherein the heavy chain has the variable region of the heavy chain described in the first aspect of the present application.
  • the heavy chain of the antibody further comprises a heavy chain constant region.
  • the heavy chain constant region is of human, murine or rabbit origin.
  • a third aspect of the present application provides a light chain variable region of an antibody, the light chain variable region comprising the following three complementarity determining region CDRs:
  • any one of the above amino acid sequences also includes at least one (such as 1-3, can be 1-2, can be 1) that is optionally added, deleted, modified and/or substituted. 1) amino acids and are able to retain IL-36R binding affinity derived sequences.
  • the light chain variable region further comprises a human FR region or a murine FR region.
  • the light chain variable region has the amino acid sequence set forth in SEQ ID NO:2.
  • the light chain variable region has the amino acid sequence set forth in SEQ ID NO: 10, 11 or 12.
  • the fourth aspect of the present application provides a light chain of an antibody, the light chain has the light chain variable region described in the third aspect of the present application.
  • the light chain of the antibody further comprises a light chain constant region.
  • the light chain constant region is of human, murine or rabbit origin.
  • a fifth aspect of the present application provides an antibody having:
  • the antibody has: a heavy chain as described in the second aspect of the present application; and/or a light chain as described in the fourth aspect of the present application.
  • the affinity constant KD(M) of the antibody binding to human IL-36R protein (for example, can be wild type) is (0.5-10) ⁇ 10 -11 , and can be (1-6) ⁇ 10 -11 , which can be (1-2) ⁇ 10 -11 .
  • the affinity constant KD(M) of the antibody for binding to human IL-1Rrp2 is (0.5-10) ⁇ 10 -11 , which can be (1-6) ⁇ 10 -11 , which can be ( 1-2) ⁇ 10 -11 .
  • the antibody is capable of blocking the binding of IL-36 ⁇ , IL-36 ⁇ , IL-36 ⁇ to IL-36R.
  • the antibody can block the secretion of cytokines mediated by IL-36 ⁇ , IL-36 ⁇ , and IL-36 ⁇ , and the cytokines can include: IL-6, IL-8, GM- CSF.
  • the antibody is selected from the group consisting of animal-derived antibodies, chimeric antibodies, humanized antibodies, or a combination thereof.
  • the antibody is a diabody, or a single-chain antibody.
  • the antibody is a monoclonal antibody.
  • the antibody is a partially or fully humanized monoclonal antibody.
  • the heavy chain variable region sequence of the antibody is set forth in SEQ ID NO: 1 or 9; and/or
  • the light chain variable region sequence of the antibody is shown in SEQ ID NO: 2, 10, 11 or 12.
  • the heavy chain variable region sequence of the antibody is set forth in SEQ ID NO:1; and the light chain variable region sequence of the antibody is set forth in SEQ ID NO:2.
  • the heavy chain variable region sequence of the antibody is set forth in SEQ ID NO: 9; and the light chain variable region sequence of the antibody is set forth in SEQ ID NO: 10, 11 or 12 .
  • the antibody is in the form of a drug conjugate.
  • the sixth aspect of the present application provides a recombinant protein, the recombinant protein has:
  • variable region of the heavy chain according to the first aspect of the present application (i) the variable region of the heavy chain according to the first aspect of the present application, the heavy chain according to the second aspect of the present application, the variable region of the light chain according to the third aspect of the present application, the fourth aspect of the present application.
  • the light chain of the aspect, or the antibody of the fifth aspect of the application (i) the variable region of the heavy chain according to the first aspect of the present application, the heavy chain according to the second aspect of the present application, the variable region of the light chain according to the third aspect of the present application, the fourth aspect of the present application.
  • the tag sequence includes a 6His tag.
  • the recombinant protein comprises a fusion protein.
  • the recombinant protein is a monomer, dimer, or multimer.
  • a seventh aspect of the present application provides a CAR construct, wherein the scFV segment of the monoclonal antibody antigen-binding region of the CAR construct is a binding region that specifically binds to IL-36R, and the scFv has the The heavy chain variable region of the first aspect of the application and the light chain variable region of the third aspect of the application.
  • the eighth aspect of the present application provides a recombinant immune cell expressing the exogenous CAR construct as described in the seventh aspect of the present application.
  • the immune cells are selected from the group consisting of NK cells, T cells.
  • the immune cells are derived from a human or non-human mammal (eg, a mouse).
  • a ninth aspect of the present application provides an antibody-drug conjugate, the antibody-drug conjugate comprising:
  • a conjugation moiety conjugated to the antibody moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.
  • the antibody moiety is coupled to the coupling moiety via a chemical bond or linker.
  • a tenth aspect of the present application provides a use of an active ingredient selected from the group consisting of the heavy chain variable region described in the first aspect of the present application, the heavy chain variable region described in the second aspect of the present application. chain, light chain variable region as described in the third aspect of the present application, light chain as described in the fourth aspect of the present application, or antibody as described in the fifth aspect of the present application, recombinant as described in the sixth aspect of the present application.
  • the protein, the immune cell according to the eighth aspect of the present application, the antibody drug conjugate according to the ninth aspect of the present application, or a combination thereof, the active ingredient is used for (a) preparing a detection reagent or kit; and /or (b) preparing a medicament for preventing and/or treating IL-36-related diseases.
  • the IL-36-related disease is an inflammatory disease mediated by IL-36.
  • the IL-36-related disease is a disease caused by hyperstimulation or mutation of the IL-36 cytokine.
  • the IL-36-related disease is selected from the group consisting of inflammation, an autoimmune disease, or a combination thereof; for example, it can be an autoimmune disease.
  • the IL-36-related disease is selected from the group consisting of psoriasis, scleroderma, chronic kidney disease, inflammatory bowel disease, psoriatic arthritis, ankylosing spondylitis, multiple sclerosis Symptoms, inflammatory arthritis, asthma, allergies, or a combination thereof, such as can be psoriasis.
  • the psoriasis includes psoriasis vulgaris, erythrodermic psoriasis, arthrotic psoriasis, generalized pustular psoriasis (GPP), palmoplantar psoriasis Pustular psoriasis (PPP).
  • the medicament is used to block the binding of IL-36 ⁇ , IL-36 ⁇ , IL-36 ⁇ to IL-36R.
  • the medicament is used to block IL-36 ⁇ , IL-36 ⁇ , IL-36 ⁇ mediated cytokine secretion.
  • the antibody is in the form of a drug conjugate (ADC).
  • ADC drug conjugate
  • the detection reagent or kit is used to diagnose IL-36-related diseases.
  • the detection reagent or kit is used to detect IL-36R protein in a sample.
  • the detection reagent is a detection sheet.
  • An eleventh aspect of the present application provides a pharmaceutical composition comprising:
  • an active ingredient selected from the group consisting of the heavy chain variable region as described in the first aspect of the present application, the heavy chain as described in the second aspect of the present application, the heavy chain as described in the third aspect of the present application
  • the pharmaceutical composition is a liquid formulation.
  • the pharmaceutical composition is an injection.
  • a twelfth aspect of the present application provides a polynucleotide encoding a polypeptide selected from the group consisting of:
  • a thirteenth aspect of the present application provides a vector containing the polynucleotide according to the twelfth aspect of the present application.
  • the vectors include bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.
  • a fourteenth aspect of the present application provides a genetically engineered host cell, wherein the host cell contains the vector described in the thirteenth aspect of the present application or the genome of the present application is integrated with the twelfth aspect of the present application. polynucleotides.
  • a fifteenth aspect of the present application provides a method for in vitro detection (including diagnostic or non-diagnostic) of IL-36R protein in a sample, the method comprising the steps of:
  • the method is non-diagnostic and non-therapeutic.
  • a sixteenth aspect of the present application provides a detection plate, the detection plate includes: a substrate (support plate) and a test strip, wherein the test strip contains the antibody as described in the fifth aspect of the application or as described in the fifth aspect of the application.
  • the antibody drug conjugate described in the ninth aspect of the present application is not limited to: a substrate (support plate) and a test strip, wherein the test strip contains the antibody as described in the fifth aspect of the application or as described in the fifth aspect of the application.
  • a seventeenth aspect of the present application provides a kit comprising:
  • the kit contains the detection plate according to the sixteenth aspect of the present application.
  • An eighteenth aspect of the present application provides a method for preparing a recombinant polypeptide, the method comprising:
  • the nineteenth aspect of the present application provides a method for preventing and/or treating IL-36-related diseases, the method comprising: administering the antibody according to the fifth aspect of the present application, the antibody of the antibody to a subject in need thereof An antibody-drug conjugate, or a CAR-T cell expressing the antibody, or a combination thereof.
  • the method further comprises: administering to the subject in need thereof other drugs or therapeutic methods for combined therapy.
  • Figure 1 shows the results of humanized candidate antibody bioactivity assay-cytokine IL-6 release.
  • Figure 2 shows the results of the humanized candidate antibody bioactivity assay-cytokine IL-8 release.
  • Figure 3 shows the results of humanized candidate antibody bioactivity assay-cytokine GM-CSF release.
  • Figure 4 shows the results of humanized candidate antibody bioactivity assay-NF- ⁇ B phosphorylation.
  • Figure 5 shows the cell-binding activity of candidate antibodies and human IL-36R.
  • Figure 6 shows the cell-binding activity of candidate antibodies and monkey IL-36R.
  • Figure 7 shows the inhibitory effect of candidate antibodies on the function of huIL-36 stimulator in NCI/ADR-RES cells.
  • Figure 8 shows the inhibitory effect of candidate antibodies on the function of huIL-36 stimulator in HIF cells.
  • Figure 9 shows the inhibitory effect of candidate antibodies on the function of huIL-36 stimulator in HIF cells.
  • Figure 10 shows the inhibitory effect of candidate antibodies on the function of huIL-36 stimulator in HIF cells.
  • Figure 11 shows the effect of candidate antibodies on imiquimod-induced psoriasis clinical score (PASI) in cynomolgus monkeys.
  • Figure 12 shows the effect of candidate antibodies on imiquimod-induced psoriasis cynomolgus monkey skin tissue HE staining pathological scores.
  • the applicant unexpectedly obtained an IL-36R antibody with high affinity and high biological activity.
  • the IL-36R antibody of the present application can bind to IL-36R with high affinity, and can block the binding of IL-36R ligands ( ⁇ , ⁇ , ⁇ ) to IL-36R, and block the activation of IL-36R ligands. signaling pathways to treat and/or prevent IL-36-related diseases. This application is completed on this basis.
  • administer refers to the application of an exogenous drug, therapeutic agent, diagnostic agent, or composition to an animal, human, subject, cell, tissue, organ, or biological fluid.
  • administering can refer to therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Treatment of cells includes contact of reagents with cells, as well as contact of reagents with fluids, and contact of fluids with cells.
  • administering and “treating” also mean in vitro and ex vivo treatment by an agent, diagnostic, binding composition, or by another cell.
  • Treatment when applied to a human, animal, or research subject, refers to therapeutic treatment, prophylactic or preventive measures, research and diagnosis; Contact of compartments or physiological fluids.
  • treating refers to the administration of an internal or external therapeutic agent, comprising any one of the IL-36R antibodies and compositions thereof of the present application, to a patient having one or more disease symptoms for which the Therapeutic agents have a therapeutic effect on these symptoms.
  • a patient is administered to a patient in an amount of the therapeutic agent effective to alleviate one or more symptoms of the disease (therapeutically effective amount).
  • the terms “optional” or “optionally” mean that the subsequently described event or circumstance can, but need not, occur.
  • “optionally comprising 1-3 antibody heavy chain variable regions” means that the antibody heavy chain variable region of a specific sequence may, but does not necessarily have, one, two or three.
  • antibody refers to an immunoglobulin, a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds.
  • the amino acid composition and sequence of the immunoglobulin heavy chain constant region are different, so their antigenicity is also different. Accordingly, immunoglobulins can be divided into five classes, or different types of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and the heavy chain constant regions corresponding to the different classes of immunoglobulins are called ⁇ , respectively. , ⁇ , ⁇ , ⁇ , and ⁇ . IgG represents the most important class of immunoglobulins.
  • Light chains are classified into kappa or lambda chains by differences in the constant region.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those in the art.
  • variable region The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly, which is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region (C region).
  • the variable region includes 3 hypervariable regions (HVR) and 4 FR regions (FR) with relatively conserved sequences.
  • HVR hypervariable regions
  • FR FR regions
  • the amino acid sequences of the four FRs are relatively conservative and do not directly participate in the binding reaction.
  • Three hypervariable regions determine the specificity of antibodies, also known as complementarity determining regions (CDRs).
  • Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of 3 CDR regions and 4 FR regions, and the sequence from the amino terminus to the carboxy terminus is FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDR regions of the light chain namely the light chain hypervariable region (LCDR), refer to LCDR1, LCDR2 and LCDR3;
  • the three CDR regions of the heavy chain namely the heavy chain hypervariable region (HCDR), refer to HCDR1, HCDR2 and HCDR3.
  • the number and position of CDR amino acid residues in the LCVR and HCVR regions of the antibody or antigen-binding fragment of the invention conform to the known Kabat numbering rules (LCDR1-3, HCDR2-3), or the numbering rules of Kabat and Chothia (HCDR1). ).
  • the four FR regions in the variable regions of native heavy and light chains are roughly in a ⁇ -sheet configuration, connected by three CDRs that form a linking loop, and in some cases can form part of a ⁇ -sheet structure.
  • the CDRs in each chain are brought together by the FR regions and together with the CDRs of the other chain form the antigen-binding site of the antibody.
  • Which amino acids make up the FR or CDR regions can be determined by comparing the amino acid sequences of antibodies of the same type.
  • the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as involvement in antibody-dependent cytotoxicity of the antibody.
  • the term "antigen-binding fragment” refers to a Fab fragment, a Fab' fragment, an F(ab')2 fragment, or a single Fv fragment having antigen-binding activity.
  • Fv antibodies contain antibody heavy chain variable regions, light chain variable regions, but no constant regions, and are the smallest antibody fragment with all antigen-binding sites.
  • Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding.
  • antigenic determinant refers to a discrete site in three-dimensional space on an antigen that is recognized by an antibody or antigen-binding fragment of the present application.
  • the present application includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Accordingly, the present application also includes fragments, derivatives and analogs of said antibodies.
  • antibodies include murine, chimeric, humanized or fully human antibodies prepared using techniques well known to those skilled in the art.
  • Recombinant antibodies such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be prepared using recombinant DNA techniques well known in the art.
  • the term "monoclonal antibody” refers to a cloned secreted antibody derived from a single cell. Monoclonal antibodies are highly specific, directed against a single epitope.
  • the cells may be eukaryotic, prokaryotic or phage clonal cell strains.
  • chimeric antibody is an antibody molecule expressed by a murine antibody V region gene and a human antibody C region gene spliced into a chimeric gene, which is then inserted into a vector and transfected into a host cell. It not only retains the high specificity and affinity of the parental mouse antibody, but also enables its human Fc segment to effectively mediate biological effector functions.
  • humanized antibody is a variable region engineered form of the murine antibody of the present application, having a variable region derived from (or substantially derived from) a non-human antibody (which may be, for example, a mouse monoclonal antibody). CDR regions, and FR regions and constant regions substantially derived from human antibody sequences; that is, grafting of murine antibody CDR region sequences onto different types of human germline antibody framework sequences. Because the CDR sequences are responsible for most antibody-antigen interactions, expression vectors can be constructed to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies.
  • antibodies can be monospecific, bispecific, trispecific, or more multispecific.
  • the antibody of the present application also includes its conservative variant, which means that compared with the amino acid sequence of the antibody of the present application, there are at most 10, may be at most 8, may be at most 5, may be at most 3 Amino acids are replaced by amino acids with similar or similar properties to form polypeptides. These conservatively variant polypeptides are best produced by amino acid substitutions according to Table A.
  • IL-36R generally refers to native or recombinant human IL-36R, as well as non-human homologues of human IL-36R. Molar concentrations of IL-36R were calculated using the molecular weight of the homodimer of IL-36R unless otherwise indicated.
  • the present application provides a highly specific and high affinity antibody against IL-36R, comprising a heavy chain and a light chain, the heavy chain containing a heavy chain variable region (VH) amino acid sequence, and the light chain containing a light chain Variable region (VL) amino acid sequences.
  • VH heavy chain variable region
  • VL light chain Variable region
  • the CDRs of the heavy chain variable region (VH) are selected from the group consisting of:
  • the CDRs of the light chain variable region (VL) are selected from the group consisting of:
  • any one of the amino acid sequences in the above-mentioned amino acid sequence also includes addition, deletion, modification and/or substitution of at least one (such as 1-5, 1-3, can be 1-2, can be 1) amino acid.
  • Derivative sequences with IL-36R binding affinity are also included in the above-mentioned amino acid sequence.
  • sequence homology formed by the addition, deletion, modification and/or substitution of at least one amino acid sequence can be at least 80%, can be at least 85%, can be at least 90%, can be is at least 95% amino acid sequence.
  • the antibody of the present application can be a double-chain or single-chain antibody, and can be selected from animal-derived antibodies, chimeric antibodies, humanized antibodies, can be humanized antibodies, human-animal chimeric antibodies, and can be fully humanized Antibody.
  • the antibody derivatives described in this application can be single-chain antibodies, and/or antibody fragments, such as: Fab, Fab', (Fab') 2 or other known antibody derivatives in the field, etc., as well as IgA, IgD, IgE , IgG and IgM antibodies or any one or more of other subtypes of antibodies.
  • the animal can be a mammal, such as a mouse.
  • the antibodies of the present application may be murine antibodies, chimeric antibodies, humanized antibodies, CDR-grafted and/or modified antibodies targeting human IL-36R.
  • VH CDR1, CDR2, CDR3 are each independently selected from any one or more sequences of SEQ ID NOs: 3, 4 and 5, or they have been added, deleted, modified and/or substituted by at least one amino acid
  • VL CDR1, CDR2, CDR3 are independently selected from any one or several sequences in SEQ ID NO: 6, 7 and 8, or they have been added, deleted, modified and/or A sequence with IL-36R binding affinity substituted for at least one amino acid.
  • the number of amino acids added, deleted, modified and/or substituted can be no more than 40% of the total number of amino acids in the initial amino acid sequence, can be no more than 35%, can be 1-33%, It can be 5-30%, it can be 10-25%, it can be 15-20%.
  • the number of amino acids added, deleted, modified and/or substituted is usually 1, 2, 3, 4 or 5, may be 1-3, may be 1-2, may be 1 .
  • any method suitable for producing monoclonal antibodies can be used to produce the anti-IL-36R antibodies of the present application.
  • animals can be immunized with a linked or naturally occurring IL-36R homodimer or fragment thereof.
  • Appropriate methods of immunization can be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes can be used.
  • IL-36R in any suitable form can be used as an immunogen (antigen) for the production of non-human antibodies specific for IL-36R, which are screened for biological activity.
  • the priming immunogen can be full-length mature human IL-36R, including native homodimers, or peptides containing single/multiple epitopes.
  • the immunogens can be used alone or in combination with one or more immunogenicity enhancers known in the art. Immunogens can be purified from natural sources, or produced in genetically modified cells.
  • the DNA encoding the immunogen can be genomic or non-genomic in origin (eg, cDNA).
  • the DNA encoding the immunogen can be expressed using suitable genetic vectors including, but not limited to, adenoviral vectors, adeno-associated viral vectors, baculovirus vectors, plasmids, and non-viral vectors.
  • Example 1 Exemplary methods of producing the anti-human IL-36R antibodies of the present application are described in Example 1.
  • Humanized antibodies can be selected from any class of immunoglobulins, including IgM, IgD, IgG, IgA and IgE.
  • the antibody is an IgG antibody, and the IgG1 subtype is used. Optimization of the necessary constant domain sequences to produce the desired biological activity is readily accomplished by screening antibodies using the biological assays described in the Examples below.
  • any type of light chain can be used in the compounds and methods herein.
  • kappa, lambda chains or variants thereof are useful in the compounds and methods of the present application.
  • Example 2 Exemplary methods of humanizing the anti-human IL-36R antibodies of the present application are described in Example 2.
  • sequences of the DNA molecules of the antibodies or fragments thereof of the present application can be obtained by conventional techniques, such as PCR amplification or genomic library screening.
  • the coding sequences for the light and heavy chains can be fused together to form single chain antibodies.
  • recombinant methods can be used to obtain the relevant sequences in bulk. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
  • synthetic methods can also be used to synthesize the relevant sequences, especially when the fragment length is short. Often, fragments of very long sequences are obtained by synthesizing multiple small fragments followed by ligation. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art.
  • the present application also relates to vectors comprising the appropriate DNA sequences described above together with appropriate promoter or control sequences. These vectors can be used to transform appropriate host cells so that they can express proteins.
  • Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells.
  • Animal cells can include (but are not limited to): CHO-S, CHO-K1, HEK-293 cells.
  • the steps of transforming host cells with recombinant DNA described in this application can be performed using techniques well known in the art.
  • the obtained transformants can be cultured by conventional methods, and the transformants express the polypeptides encoded by the genes of the present application. Depending on the host cell used, it is cultured with conventional media under appropriate conditions.
  • the transformed host cells are cultured under conditions suitable for expression of the antibodies of the present application. Then use conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography and other techniques in the art
  • immunoglobulin purification steps such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography and other techniques in the art
  • the antibody of the present application can be obtained after purification by conventional separation and purification methods well known to those skilled in the art.
  • the resulting monoclonal antibodies can be identified by conventional means.
  • the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • ADCs Antibody-Drug Conjugates
  • the present application also provides an antibody-drug conjugate (ADC) based on the antibody of the present application.
  • ADC antibody-drug conjugate
  • the antibody-drug conjugate includes the antibody, and an effector molecule, the antibody is coupled to the effector molecule, and may be chemically coupled.
  • the effector molecule can be a drug with therapeutic activity.
  • the effector molecule can be one or more of a toxin, a chemotherapeutic drug, a small molecule drug, or a radionuclide.
  • the antibody of the present application and the effector molecule can be coupled through a coupling agent.
  • the coupling agent may be any one or more of non-selective coupling agents, coupling agents utilizing carboxyl groups, peptide chains, and coupling agents utilizing disulfide bonds.
  • the non-selective coupling agent refers to a compound that forms a covalent bond between the effector molecule and the antibody, such as glutaraldehyde and the like.
  • the coupling agent utilizing the carboxyl group may be any one or more of a cis-aconitic anhydride type coupling agent (such as cis-aconitic anhydride) and an acyl hydrazone type coupling agent (the coupling site is an acyl hydrazone).
  • antibodies are used to link with various functional groups, including imaging reagents (such as chromophores and fluorophores), diagnostic reagents (such as MRI contrast agents and radioisotopes) , stabilizers (eg, ethylene glycol polymers) and therapeutic agents.
  • imaging reagents such as chromophores and fluorophores
  • diagnostic reagents such as MRI contrast agents and radioisotopes
  • stabilizers eg, ethylene glycol polymers
  • therapeutic agents eg, ethylene glycol polymers
  • Antibodies can be conjugated to functional agents to form antibody-functional agent conjugates.
  • Functional agents eg, drugs, detection reagents, stabilizers
  • the functional agent can be attached to the antibody either directly or indirectly through a linker.
  • Antibodies can be conjugated to drugs to form antibody drug conjugates (ADCs).
  • ADC antibody drug conjugates
  • the ADC contains a linker between the drug and the antibody.
  • Linkers can be degradable or non-degradable linkers. Degradable linkers are typically susceptible to degradation in the intracellular environment, eg, at the target site, where the linker is degraded, thereby releasing the drug from the antibody.
  • Suitable degradable linkers include, for example, enzymatically degradable linkers, including peptidyl-containing linkers that can be degraded by intracellular proteases (eg, lysosomal or endosomal proteases), or sugar linkers that, for example, can be degraded by glucuronides Enzymatically degraded glucuronide-containing linkers.
  • Peptidyl linkers can include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine, or valine-alanine.
  • degradable linkers include, for example, pH sensitive linkers (eg, linkers that hydrolyze at pH less than 5.5, eg, hydrazone linkers) and linkers that degrade under reducing conditions (eg, disulfide linkers).
  • Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.
  • the linker Before being attached to the antibody, the linker has a reactive reactive group capable of reacting with certain amino acid residues, and the attachment is achieved through the reactive reactive group.
  • Sulfhydryl-specific reactive groups can include, for example, maleimides, halogenated amides (eg, iodo, bromo, or chloro); halogenated esters (eg, iodo, bromo, or chloro); halogens substituted methyl ketones (eg iodo, bromo or chloro), benzyl halides (eg iodo, bromo or chloro); vinyl sulfones, pyridyl disulfides; mercury derivatives such as 3,6-bis-( mercury methyl) dioxane, and the counter ion is acetate, chloride or nitrate; and polymethylene dimethyl sulfide thiosulfonate.
  • Linkers can include, for example, maleimide linked
  • the drug can be any cytotoxic, cytostatic or immunosuppressive drug.
  • the linker connects the antibody and the drug, and the drug has a functional group that can form a bond with the linker.
  • the drug can have an amino, carboxyl, sulfhydryl, hydroxyl, or keto group that can form a bond with the linker.
  • the drug is directly attached to the linker, the drug has a reactive reactive group prior to attachment to the antibody.
  • Useful drug classes include, for example, antitubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folate antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors , Vinca alkaloids, etc.
  • drug-linkers can be used to form ADCs in one simple step.
  • bifunctional linker compounds can be used to form ADCs in a two- or multi-step process. For example, a cysteine residue reacts with the reactive moiety of the linker in the first step, and in a subsequent step, the functional group on the linker reacts with the drug to form the ADC.
  • functional groups on the linker are selected to facilitate specific reaction with suitable reactive groups on the drug moiety.
  • azide-based moieties can be used to specifically react with reactive alkynyl groups on drug moieties.
  • the drug is covalently bound to the linker through a 1,3-dipolar cycloaddition between the azide and the alkynyl group.
  • Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines).
  • the present application also provides a method for preparing an ADC, which may further include: combining the antibody with the drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).
  • the methods of the present application comprise: binding an antibody to a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the methods of the present application further comprise: conjugating the antibody linker conjugate to the drug moiety under conditions sufficient to covalently link the drug moiety to the antibody through the linker.
  • the antibody drug conjugate ADC is represented by the following molecular formula:
  • Ab is an antibody
  • D is a drug
  • the present application provides the use of the antibody of the present application, for example, for the preparation of diagnostic preparations, or the preparation of medicaments for the prevention and/or treatment of IL-36-related diseases.
  • the IL-36-related diseases include inflammatory diseases, autoimmune diseases, etc., including but not limited to psoriasis, psoriatic arthritis, ankylosing spondylitis, multiple sclerosis, inflammatory bowel disease (such as Crohn's disease).
  • osteoarthritis e.g scleroderma, pulmonary fibrosis and sclerosis
  • inflammatory fibrosis eg scleroderma, pulmonary fibrosis and sclerosis
  • asthma including allergic asthma
  • allergies and cancer.
  • the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein or its ADC or corresponding CAR-T cells, and a pharmaceutically acceptable carrier.
  • these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, usually at a pH of about 5-8, and can be at a pH of about 6-8, although the pH may vary depending on the formulation It varies depending on the nature of the substance and the condition being treated.
  • the formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intratumoral, intraperitoneal, intravenous, or topical administration.
  • the antibody described in the present application can also be expressed in a cell by a nucleotide sequence for cell therapy, for example, the antibody is used for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.
  • CAR-T chimeric antigen receptor T cell immunotherapy
  • the pharmaceutical composition of the present application can be directly used to bind IL-36R protein molecules, and thus can be used to prevent and treat IL-36-related diseases.
  • other therapeutic agents may also be used concomitantly.
  • the pharmaceutical composition of the present application contains a safe and effective amount (eg, 0.001-99 wt %, can be 0.01-90 wt %, can be 0.1-80 wt %) of the above-mentioned monoclonal antibody (or its conjugate) of the present application and a pharmaceutically acceptable amount.
  • Accepted carrier or excipient include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the drug formulation should match the mode of administration.
  • the pharmaceutical composition of the present application can be prepared in the form of injection, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants.
  • compositions such as injections and solutions are preferably manufactured under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, eg, about 1 microgram/kg body weight to about 5 mg/kg body weight per day.
  • the polypeptides of the present application can also be used with other therapeutic agents.
  • a safe and effective amount of the pharmaceutical composition is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 mg per kilogram of body weight, and can be The dosage for this is about 10 micrograms/kg body weight to about 20 mg/kg body weight.
  • the specific dosage should also take into account the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.
  • the antibodies of the present application can be used in detection applications, eg, in detection of samples, to provide diagnostic information.
  • sample used includes cells, tissue samples and biopsy specimens.
  • biopsy shall include all kinds of biopsies known to those skilled in the art. Biopsy as used in this application may thus include tissue samples prepared, for example, by endoscopic methods or needle or needle biopsy of an organ.
  • Samples used in this application include fixed or preserved cell or tissue samples.
  • the present application also provides a kit containing the antibody (or its fragment) of the present application, for example, the kit may further include a container, instructions for use, buffers, and the like.
  • the antibodies of the present application can be immobilized on a detection plate.
  • the antibody of the present application has excellent biological activity and specificity.
  • the antibody of the present application has an affinity for IL-36R of some non-human mammals (such as monkeys) that is comparable to human IL-36R, which is convenient for testing in animal models and for quality control detection.
  • the antibody of this application blocks the activation signal pathway of IL-36 ⁇ , IL-36 ⁇ , IL-36 ⁇ and IL-36R by binding to IL-36R, and reduces inflammatory cytokines (such as IL-6, IL-8, GM) -CSF)
  • the method for preparing murine monoclonal antibody adopts the hybridoma preparation technology invented by Kohler and Milstein in 1975 (Nature, 1975, 256: 495-497).
  • the human IL-36R Fc-tagged protein (ACRO, #IL2-H5254) was first emulsified with Freund's adjuvant and then multiple strains of mice were immunized. After four rounds of immunization, serum was taken to detect the titer by ELISA, and splenocytes from the best mice were selected for fusion with SP2/0 myeloma cells.
  • hybridoma polyclonal cells were screened by HAT, and the supernatant of polyclonal culture was tested for the binding activity of human IL-36R, monkey IL-36R, and human IL1R1, as well as the functional inhibition of polyclonal antibodies to IL-36 stimulator.
  • the best polyclonal clones were selected for monoclonalization, the binding activity and functional activity of the monoclones were also screened, and the affinity was detected by the Biacore method. Finally, the best hybridoma monoclonal cell lines were selected for sequence analysis.
  • hybridomas 6(1)-B8 and 111(1)-E5 antibodies expressed high levels of cell binding to human IL-36R and monkey IL-36R proteins. It has no specific binding to human IL1R1 protein, and has obvious functional blocking effect on cytokines stimulated by IL-36 stimulators (IL-36 ⁇ , IL-36 ⁇ , IL-36 ⁇ ).
  • variable region gene-specific cDNAs for heavy and light chains were synthesized using the SMARTer 5'RACE Synthesis Kit (TAKARA, Cat. No. 634859) according to the manufacturer's instructions.
  • the 5' and 3' ends of the cDNA sequence are modified with PCR primers designed to add appropriate leader sequences to the heavy and light chain variable region cDNAs, respectively, so that the resulting PCR products can be cloned into The heavy chain carrier pHB-Fc and the light chain carrier pHB-CK are expressed on the existing recombinant antibody.
  • the pHB-Fc expression vector contains the human IgG1 heavy chain constant region gene sequence, and CH2 has L234A and L235A (Eu numbering) mutations that weaken the antibody ADCC effect; the pHB-C ⁇ vector contains the human kappa light chain constant region gene sequence.
  • the PCR amplification products of the variable regions of the heavy and light chains were cloned into an expression vector by In-fusion cloning reagent (TAKARA, Cat. No. 639650) to obtain a human-mouse chimeric antibody expression vector, which was transformed into E.coli DH5 ⁇ Escherichia coli for sensing Live cells (Probiotics, Cat. No. FYE607-80VL).
  • Antibody variable region sequences were obtained by selecting monoclonal colonies for Sanger sequencing.
  • an anti-IL-36R chimeric antibody (Huabo No. 900497) was obtained, the light chain of which was derived from hybridoma 6(1)-B8 in Example 1, and the heavy chain was derived from hybridoma 111(1) in Example 1 -E5, whose variable region sequence is as follows:
  • CDRs IMGT definition, a single column is as follows:
  • the expression vector obtained in 2.1 was amplified by E. coli, and a sufficient amount of plasmid was prepared with an endotoxin-removing plasmid extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., Item No. DP117) for transient transfection and expression of chimeric antibodies.
  • the host cells used for expression were CHO-S cells (Thermo Fisher, Cat. No. R80007).
  • the prepared two heavy chain carriers were mixed with the light chain carrier and polyetherimide (PEI, Polysciences, Cat. No. 24765-1) to form a liposome complex, and then transfected into CHO-S cells and put Incubate in a carbon dioxide shaker for 5-7 days.
  • the cell culture supernatant was collected by centrifugation, and purified by Protein A affinity chromatography to obtain a human-mouse chimeric antibody.
  • the humanization of the antibody adopts the following method:
  • the variable region sequence of the chimeric antibody (Huabo No. 900497) is compared with the available sequences in the NCBI IgBlast database, and through identification and analysis, it is finally determined that it is suitable for the construction of CDR grafts on it.
  • the transformation site is designed, and the variable regions of the heavy and light chains of the chimeric antibody are respectively designed for humanization mutation.
  • PCR technology to amplify and construct humanized point mutant antibody expression plasmid.
  • the humanized point mutant antibody expression plasmids were expressed in CHO-S cells respectively, and the humanized antibody protein was obtained after purification.
  • Biacore and cell biological activity detection methods the affinity of humanized antibodies, activated cytokine release and other indicators were screened, and three humanized anti-IL-36R antibodies with excellent performance were obtained.
  • the obtained humanized anti-IL-36R antibodies are numbered 900513, 900527 and 900534, and their VH and VL sequences are shown below:
  • the underlined regions are CDRs (defined by IMGT), and 900513, 900527 and 900534 are the three humanized antibody protein numbers, respectively.
  • the CDR regions of the humanized antibodies 900513, 900527 and 900534 are the same as the CDR regions of the chimeric antibody 900497, and the FR region of the humanized antibody is mutated on the basis of the chimeric 900497.
  • test materials and instruments are as follows:
  • IL-1 Rrp2/IL-1 R6 protein Human IL-1 Rrp2/IL-1 R6 Protein, His Tag
  • ACRO Biosystems ACRO Biosystems, IL2-H52H6
  • the test method is as follows:
  • Sereis S Sensor Chip CM5 chip was equilibrated at room temperature for 20-30min, and the chip was loaded into the instrument.
  • the anti-His antibody was immobilized on the Series S Sensor Chip CM5 chip according to the instructions of the His Capture Kit.
  • the HBS-EP+(10X) solution was diluted 10 times with ultrapure water as the Running Buffer.
  • the antigen His-tagged human IL-1 Rrp2/IL-1 R6 protein
  • 900389 is an anti-human IL-36R antibody developed by BI. It was synthesized by Huabo according to the sequence recorded in the patent (WO2013/074569A1), and the variable region gene was independently expressed. Specifically, the heavy chain variable region of 900389 is 81B4vH33_90vH shown in SEQ ID NO:89 in WO2013/074569A1, and the light chain variable region of 900389 is 81B4vK32_105vK, 81B4vH33_905vK2_105vK2_105vK2_105vK2_105vK2_105vK2_105vK2_105vK2_105vK and 81B4vK shown in SEQ ID NO:77 in WO2013/074569A1 All were obtained by humanization transformation on the basis of murine antibody 81B4; the constant region of 900389 is the same as the candidate antibody of this application.
  • Table 3 shows the affinity constant (KD(M)) of each humanized antibody binding to human IL-1 Rrp2. The results show that the affinity of the applicant's humanized monoclonal antibody is close to the order of 10 -11 , and has a very strong affinity.
  • the human ovarian cancer cell line NCI/ADR-RES was added to a 96-well cell culture plate at 100 ⁇ L per well (4.5 ⁇ 10 4 cells per well), incubated overnight at 37°C, 5% CO 2 , and the samples to be tested were serially diluted with 50 ⁇ L Add/well, incubate at 37°C, 5% CO 2 for 15 min, add ligands huIL-36 ⁇ (1 ⁇ g/mL), huIL-36 ⁇ (80 ng/mL), huIL-36 ⁇ (120 ng/mL) at 50 ⁇ L/well, respectively, After thorough mixing, incubate at 37°C, 5% CO 2 for 18-24h.
  • the cell culture supernatant was collected by centrifugation, and detected with Biolegend ELISA kit: huIL-6 (1:100 dilution, Cat. No. 430503), huIL-8 (1:5 dilution, Cat. No. 431503) and huGM-CSF (1:2 dilution, Cat. No. 431503) 432003).
  • Detect the activation of NF- ⁇ B in NCI/ADR-RES cells under the stimulation of huIL-36 ⁇ Dilute the ligand huIL-36 ⁇ to 240ng/mL, add it to the cells pre-incubated with the antibody to be tested, 37°C, 5 % CO2 for 30min. The supernatant was removed by centrifugation, the cells were lysed, and the lysate was collected for NF- ⁇ B phosphorylation detection using a kit (Cisbio, Cat. No. 64NFBPEG).
  • the humanized antibodies 900513, 900527, 900534 have a significant blocking effect on the functions of human stimulatory factors IL-36 ⁇ , IL-36 ⁇ , IL-36 ⁇ , and can significantly block The secretion of cytokines IL-6, IL-8, GM-CSF and the phosphorylation of NF- ⁇ B were interrupted.
  • the Series S Sensor Chip CM5 (GE, #BR-1005-30) was equilibrated at room temperature for 20-30 min, and then loaded into a Biacore 8K (GE) instrument. Egg-derived lysozyme solution (Sigma, #L3790) and soybean-derived trypsin inhibitor type 1-S (Sigma, #T-2327) were separated using an amino coupling kit (GE, #BR-1000-50). Fixed to CM5 chip.
  • the injection buffer was HBS-EP (1X) (GE, #BR-1006-69), and 4 equilibration cycles were set.
  • Polyclonal rabbit anti-lysozyme (ABcam, Ab391), anti-trypsin inhibitor antibody (Anti-trypsin inhibitor antibody, LifeSpan Biosciences, #LS-C76609), chimeric antibody and humanized antibody were diluted with equilibration buffer to 1000nM, set flow rate 5 ⁇ L/min, injection channel 1, 2 and 3, Flow Cell 1 and 2.
  • the binding time was 10 min, and the dissociation time was 15 min.
  • the regeneration flow rate was 50 ⁇ L/min, and the regeneration was performed with 0.85% phosphoric acid solution (ProteOn, 176-2260) for 60 s, and then with 50 mM sodium hydroxide solution for 30 s.
  • sample 900389 is an anti-human IL-36R humanized antibody developed by BI, which was independently expressed by Huabo synthesizing the variable region gene according to the patent.
  • Sample 900497 is an anti-IL-36R chimeric antibody constructed by Huabo hybridoma screening.
  • the BI control sequence 900389, the humanized sequence 900527 screened by Huabo, and TNP IgG1 (Fc silence) 900543 were inserted into Huabo GS vector, respectively, to construct a recombinant expression plasmid and transfected into CHO-K1 cells to construct stable cell lines.
  • the protein number of the stable cell line constructed by Huabo 900527 is HB0034.
  • the selected monoclonal cell line has an expression level of more than 4g/L after being optimized by the upstream process.
  • the antibodies expressed by the stably transfected cell lines were purified and tested by flow cytometry binding experiments and functional experiments.
  • Sample 900543 is a TNP IgG1 (Fc silence) negative control antibody.
  • the antibody of the stable cell line was tested for the binding activity of human IL-36R and monkey IL-36R, and the flow detection cell line was the CHO-K1 cell line constructed by Huabo that expresses human IL-36R and monkey IL-36R.
  • Huabo monoclonal antibody HB0034 had high binding activity to human IL-36R and monkey IL-36R.
  • the antibody of the present application has a lower EC50 value and has stronger binding activity to human IL-36R.
  • the human ovarian cancer cell line NCI/ADR-RES was added to a 96-well cell culture plate at 100 ⁇ L per well (4.5 ⁇ 10 4 cells per well), incubated overnight at 37°C, 5% CO 2 , and the samples to be tested were serially diluted with 50 ⁇ L Add/well, incubate at 37°C, 5% CO2 for 15min, add ligand huIL-36 ⁇ (40ng/mL) at 50 ⁇ L/well, mix well, incubate at 37°C, 5% CO2 for 18-24h. The cell culture supernatant was collected by centrifugation, and huIL-6 was detected by HTRF kit (Cat#62HIL06PEG, CISBIO).
  • the stable cell line humanized antibody HB0034 has a significant blocking effect on the function of the human stimulatory factor IL-36 ⁇ , and can significantly block the secretion of the cytokine IL-6.
  • the antibody of the present application has a lower IC50 value and a stronger blocking effect.
  • HIF Human Intestinal Fibroblasts
  • Serum Starvd medium 100ul (45000 cells) per well was added with 96wp, and incubated overnight. After serial dilution of the antibody, 50ul/well was added and incubated at 37°C for 15min.
  • IL-36 ⁇ was diluted to 1000ng/ml, 50ul was added to each well, and mixed well. Incubate for 4 h at 37°C in 5% CO 2 , collect the supernatant by centrifugation, and detect the content of human IL-6.
  • IL-36 ⁇ was diluted to 80ng/m, and 50ul was added to each well, and mixed well.
  • the stable cell line humanized antibody HB0034 has a significant blocking effect on the functions of human stimulatory factors IL-36 ⁇ and IL-36 ⁇ , and can significantly block the secretion of the cytokine IL-6 and the phosphorylation of NF- ⁇ B. .
  • the antibody of the present application has a lower IC50 value and a stronger blocking effect.
  • the Series S Sensor Chip CM5 (GE, #BR-1005-30) was equilibrated at room temperature for 20-30 minutes, and then loaded into a Biacore 8K (GE) instrument. Lysozyme solution (Sigma, #L3790) from egg and trypsin inhibitor type 1-S (Sigma, #T-2327) from soybean were separated using an amino coupling kit (GE, #BR-1000-50). Fixed to CM5 chip channels 1 and 2. The running buffer was HBS-EP+(1X) (GE, #BR-1006-69), and 5 equilibration cycles were set.
  • Polyclonal rabbit anti-lysozyme (ABcam, #Ab391), Anti-trypsin inhibitor antibody (LifeSpan Biosciences, #LS-C76609), chimeric antibody and humanized antibody were diluted to 1000nM with running buffer, set flow rate 5 ⁇ L/min , injection channel 1, 2 and 3, Flow Cell 1 and 2. The binding time was 10 min, and the dissociation time was 15 min. The regeneration flow rate was 50 ⁇ L/min. First, 0.85% phosphoric acid solution (BIO-RAD, #176-2260) was used for regeneration for 60s, and then 50mM sodium hydroxide solution (GE, #BR-1003-58) was used for regeneration for 30s.
  • BIO-RAD 0.85% phosphoric acid solution
  • GE 50mM sodium hydroxide solution
  • Example 7 Therapeutic effect of humanized antibody on psoriasis
  • the cynomolgus monkeys were divided into 5 groups with 6 rats in each group, namely model control group, positive control group, HB0034 low-dose group, HB0034 medium-dose group and HB0034 high-dose group.
  • Imiquimod cream (IMQ) was applied to form a psoriasis model. Beginning on the day of the first modeling (Day0) and on the fourth day (Day4), HB0034 low-dose, medium-dose and high-dose groups were given 5, 15, and 50 mg/kg HB0034 intravenously, respectively, the model control group was given normal saline, and the positive control group was given groundwater Dexamethasone (5 mg/ cm2 , administered as a smear, once daily).
  • IL-17 and IL-36 in the skin were significantly decreased (p ⁇ 0.05) (see Table 15, Figures 11-12).
  • HB0034 at doses of 15-50 mg/kg can dose-dependently improve IMQ-induced skin erythema, dandruff and psoriasis-like symptoms of skin thickening, reduce PASI score, improve The skin pathological score of the modeling site and the reduction of IL-17 and IL-36 levels in skin tissue.
  • the minimum effective dose of HB0034 was 15 mg/kg.

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Abstract

本申请提供了靶向于白介素36R的抗体及其制备方法和应用。具体地，本申请提供了高亲和力高生物活性的IL-36R抗体，所述抗体能够与IL-36R高亲和力结合，并且能够阻断IL-36R配体(α、β、γ)与IL-36R结合，阻断IL-36R配体激活的信号通路，从而治疗和/或预防IL-36相关疾病。

Description

靶向于白介素36R的抗体及其制备方法和应用 技术领域
本申请涉及生物医药领域，具体的涉及一种靶向于白介素36R的抗体及其制备方法和应用。
背景技术
IL-1受体家族成员(IL1R)包含至少11个成员，其中包括IL1RI(IL1R1)、IL1RII(IL1R2)、IL1RAcP(IL1R3)、ST2(T1/IL1R4)、IL18Ra(IL1Rrp/IL1R5)、IL1Rrp2(IL1RL2/IL1R6/IL-36R)、IL18Rb(AcPL/IL1R7)、IL1RAPL1(TIGIRR2/IL1R8)、和TIGIRR1(IL1R9)。IL-36R可识别3个不同的激活配体细胞因子，IL36α，IL36β，IL36γ(也被称为IL-1F6、IL-1F8、IL-1F9)，引发炎症细胞因子的表达。并有2个天然拮抗剂IL-36Ra(IL-1F5)和IL-38,他们与IL-36R结合后会抑制炎症细胞因子的表达。
IL-36R在肺上皮细胞、脑血管细胞、肾、睾丸、单核细胞、皮肤衍生的角质细胞、成纤维细胞和内皮细胞上表达。IL-36R由细胞膜外配体结合结构域、跨膜螺旋和细胞内信号转导Toll/IL-1受体结构域(Toll/IL-1receptor domain，TIR结构域)组成。IL-36α、IL-36β、IL-36γ首先与IL-36R结合，使其与IL-1RAcP形成信号转导复合体，然后募集髓样分化因子88(myeloid differentiation factor 88，MyD88)，激活由c-Jun氨基末端激酶(c-Jun N-terminal kinases，JNK)和细胞外调节蛋白激酶(extracellular regulated protein kinases，ERK1/2)介导的丝裂原激活蛋白激酶(mitogen-activated protein kinases，MAPK)和核因子Kappa B(nuclear factor kappa B，NF-κB)途径，产生大量炎症介质，进而介导炎症反应，在适应性免疫中发挥重要作用。IL-36Ra与IL-36R的结合不会导致IL-1RAcP的募集，促炎性级联反应未开启，实现了IL-36Ra的抗炎特性。在正常人体中，IL-36α、IL-36β、IL-36γ介导的信号激活作用和IL-36Ra介导的信号抑制作用为平衡状态，当IL-36Ra发生基因突变时，会导致其抑制功能失活，打破平衡状态，发生炎症疾病。
IL-36细胞因子参与了某些严重的银屑病，包括泛发性脓疱性银屑病(GPP)和掌跖脓疱病(PPP)。GPP是一种严重的全身型类型脓疱型银屑病，具有致死性。PPP是慢性的影响手掌和脚掌的脓疱型银屑病。目前针对GPP和PPP的治疗方法包括口服类视黄醇和外用类固醇，但是治疗疗效差并有严重副作用。
因此，本领域仍需要能够与IL-36R高亲和力结合，并且能够阻断IL-36α、IL-36β、IL-36γ与IL-36R结合的抗IL-36R抗体，用于治疗GPP、PPP等IL-36相关疾病。
发明内容
本申请的目的在于提供靶向于白介素36R的抗体及其制备方法和应用。
具体地，本申请的目的在于提供一种高亲和力高生物活性的IL-36R抗体及其应用，能够与IL-36R高亲和力结合，并且能够阻断IL-36α、IL-36β、IL-36γ与IL-36R结合。
本申请的另一目的在于提供一种白介素36R结合分子及其用途，特别是在治疗和/或预防、或诊断IL-36相关疾病如银屑病中的用途。
本申请的第一方面，提供了一种抗体的重链可变区，所述的重链可变区包括以下三个互补决定区CDR：
SEQ ID NO:3所示的CDR1，
SEQ ID NO:4所示的CDR2，和
SEQ ID NO:5所示的CDR3。
在某些实施方式中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个(如1-3个，可以为1-2个，可以为1个)氨基酸并能够保留IL-36R结合亲和力的衍生序列。
在某些实施方式中，所述重链可变区还包括人源的FR区或鼠源的FR区。
在某些实施方式中，所述重链可变区具有SEQ ID NO:1所示的氨基酸序列。
在某些实施方式中，所述重链可变区具有SEQ ID NO:9所示的氨基酸序列。
本申请的第二方面，提供了一种抗体的重链，所述的重链具有如本申请第一方面所述的重链可变区。
在某些实施方式中，所述的抗体的重链还包括重链恒定区。
在某些实施方式中，所述的重链恒定区为人源、鼠源或兔源的。
本申请的第三方面，提供了一种抗体的轻链可变区，所述的轻链可变区包括以下三个互补决定区CDR：
SEQ ID NO:6所示的CDR1’，
SEQ ID NO:7所示的CDR2’，和
SEQ ID NO:8所示的CDR3’。
在某些实施方式中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个(如1-3个，可以为1-2个，可以为1个)氨基酸并能够保留IL- 36R结合亲和力的衍生序列。
在某些实施方式中，所述轻链可变区还包括人源的FR区或鼠源的FR区。
在某些实施方式中，所述轻链可变区具有SEQ ID NO:2所示的氨基酸序列。
在某些实施方式中，所述轻链可变区具有SEQ ID NO:10、11或12所示的氨基酸序列。
本申请的第四方面，提供了一种抗体的轻链，所述的轻链具有如本申请第三方面所述的轻链可变区。
在某些实施方式中，所述的抗体的轻链还包括轻链恒定区。
在某些实施方式中，所述的轻链恒定区为人源、鼠源或兔源的。
本申请的第五方面，提供了一种抗体，所述抗体具有：
(1)如本申请第一方面所述的重链可变区；和/或
(2)如本申请第三方面所述的轻链可变区；
或者，所述抗体具有：如本申请第二方面所述的重链；和/或如本申请第四方面所述的轻链。
在某些实施方式中，所述抗体与人IL-36R蛋白(例如可以为野生型)结合的亲和力常数KD(M)为(0.5-10)×10 -11，可以为(1-6)×10 -11，可以为(1-2)×10 -11。
在某些实施方式中，所述抗体与对人IL-1Rrp2结合的亲和力常数KD(M)为(0.5-10)×10 -11，可以为(1-6)×10 -11，可以为(1-2)×10 -11。
在某些实施方式中，所述抗体能够阻断IL-36α，IL-36β，IL-36γ与IL-36R的结合。
在某些实施方式中，所述抗体能够阻断IL-36α，IL-36β，IL-36γ介导的细胞因子分泌，可以为，所述细胞因子包括：IL-6,IL-8,GM-CSF。
在某些实施方式中，所述抗体选自：动物源抗体、嵌合抗体、人源化抗体、或其组合。
在某些实施方式中，所述的抗体为双链抗体、或单链抗体。
在某些实施方式中，所述的抗体为单克隆抗体。
在某些实施方式中，所述的抗体是部分或全人源化的单克隆抗体。
在某些实施方式中，所述抗体的重链可变区序列如SEQ ID NO：1或9所示；和/或
所述的抗体的轻链可变区序列如SEQ ID NO：2、10、11或12所示。
在某些实施方式中，所述抗体的重链可变区序列如SEQ ID NO：1所示；并且所述的抗体的轻链可变区序列如SEQ ID NO：2所示。
在某些实施方式中，所述抗体的重链可变区序列如SEQ ID NO：9所示；并且所述的抗体的轻链可变区序列如SEQ ID NO：10、11或12所示。
在某些实施方式中，所述的抗体为药物偶联物形式。
本申请的第六方面，提供了一种重组蛋白，所述的重组蛋白具有：
(i)如本申请第一方面所述的重链可变区、如本申请第二方面所述的重链、如本申请第三方面所述的轻链可变区、如本申请第四方面所述的轻链、或本申请第五方面所述的抗体；以及
(ii)任选的协助表达和/或纯化的标签序列。
在某些实施方式中，所述的标签序列包括6His标签。
在某些实施方式中，所述的重组蛋白(或多肽)包括融合蛋白。
在某些实施方式中，所述的重组蛋白为单体、二聚体、或多聚体。
本申请的第七方面，提供了一种CAR构建物，所述的CAR构建物的单克隆抗体抗原结合区域的scFV段为特异性结合于IL-36R的结合区，并且所述scFv具有如本申请第一方面所述的重链可变区和如本申请第三方面所述的轻链可变区。
本申请的第八方面，提供了一种重组的免疫细胞，所述的免疫细胞表达外源的如本申请第七方面所述的CAR构建物。
在某些实施方式中，所述的免疫细胞选自下组：NK细胞、T细胞。
在某些实施方式中，所述的免疫细胞来自人或非人哺乳动物(如鼠)。
本申请的第九方面，提供了一种抗体药物偶联物，所述的抗体药物偶联物含有：
(a)抗体部分，所述抗体部分选自下组：如本申请第一方面所述的重链可变区、如本申请第二方面所述的重链、如本申请第三方面所述的轻链可变区、如本申请第四方面所述的轻链、或本申请第五方面所述的抗体、或其组合；和
(b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、或其组合。
在某些实施方式中，所述的抗体部分与所述的偶联部分通过化学键或接头进行偶联。
本申请的第十方面，提供了一种活性成分的用途，所述活性成分选自下组：如本申请第一方面所述的重链可变区、如本申请第二方面所述的重链、如本申请第三方面所述的轻链可变区、如本申请第四方面所述的轻链、或本申请第五方面所述的抗体、如本申请第六方面所述的重组蛋白、如本申请第八方面所述的免疫细胞、如本申请第九方面所述的抗体药物偶联物、或其组合，所述活性成分用于(a)制备检测试剂或试剂盒；和/或(b)制备预防和/或治疗IL-36相关疾病的药物。
在某些实施方式中，所述IL-36相关疾病是由IL-36介导的炎症疾病。
在某些实施方式中，所述IL-36相关疾病是由IL-36细胞因子的过度刺激或突变引起的疾病。
在某些实施方式中，所述IL-36相关疾病选自下组：炎症、自身免疫疾病、或其组合；例如可以为自身免疫疾病。
在某些实施方式中，所述的IL-36相关疾病选自下组：银屑病、硬皮病、慢性肾病、发炎性肠疾病、银屑病关节炎、强直性脊柱炎、多发性硬化症、炎性关节炎、哮喘、过敏、或其组合，例如可以为银屑病。
在某些实施方式中，所述的银屑病包括寻常型银屑病、红皮病型银屑病、关节病型银屑病、泛发性脓疱型银屑病(GPP)、掌跖脓疱型银屑病(PPP)。
在某些实施方式中，所述药物用于阻断IL-36α，IL-36β，IL-36γ与IL-36R的结合。
在某些实施方式中，所述药物用于阻断IL-36α，IL-36β，IL-36γ介导的细胞因子分泌。
在某些实施方式中，所述的抗体为药物偶联物(ADC)形式。
在某些实施方式中，所述的检测试剂或试剂盒用于诊断IL-36相关疾病。
在某些实施方式中，所述检测试剂或试剂盒用于检测样品中IL-36R蛋白。
在某些实施方式中，所述的检测试剂为检测片。
本申请的第十一方面，提供了一种药物组合物，所述的药物组合物含有：
(i)活性成分，所述活性成分选自下组：如本申请第一方面所述的重链可变区、如本申请第二方面所述的重链、如本申请第三方面所述的轻链可变区、如本申请第四方面所述的轻链、或本申请第五方面所述的抗体、如本申请第六方面所述的重组蛋白、如本申请第八方面所述的免疫细胞、如本申请第九方面所述的抗体药物偶联物、或其组合；以及
(ii)药学上可接受的载体。
在某些实施方式中，所述的药物组合物为液态制剂。
在某些实施方式中，所述的药物组合物为注射剂。
本申请的第十二方面，提供了一种多核苷酸，所述的多核苷酸编码选自下组的多肽：
(1)如本申请第一方面所述的重链可变区、如本申请第二方面所述的重链、如本申请第三方面所述的轻链可变区、如本申请第四方面所述的轻链、或本申请第五方面所述的抗体；或
(2)如本申请第六方面所述的重组蛋白；
(3)如本申请第七方面所述的CAR构建物。
本申请的第十三方面，提供了一种载体，所述的载体含有如本申请第十二方面所述的多核苷酸。
在某些实施方式中，所述的载体包括：细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒、或其他载体。
本申请的第十四方面，提供了一种遗传工程化的宿主细胞，所述的宿主细胞含有如本申请第十三方面所述的载体或基因组中整合有如本申请第十二方面所述的多核苷酸。
本申请的第十五方面，提供了一种体外检测(包括诊断性或非诊断性)样品中IL-36R蛋 白的方法，所述方法包括步骤：
(1)在体外，将所述样品与如本申请第五方面所述的抗体或如本申请第九方面所述的抗体药物偶联物、接触；
(2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在IL-36R蛋白。
在某些实施方式中，所述的方法是非诊断非治疗的。
本申请的第十六方面，提供了一种检测板，所述的检测板包括：基片(支撑板)和测试条，所述的测试条含有如本申请第五方面所述的抗体或如本申请第九方面所述的抗体药物偶联物。
本申请的第十七方面，提供了一种试剂盒，所述试剂盒中包括：
(1)第一容器，所述第一容器中含有如本申请第五方面所述的抗体；和/或
(2)第二容器，所述第二容器中含有抗如本申请第五方面所述的抗体的二抗；
或者，所述试剂盒含有如本申请第十六方面所述的检测板。
本申请的第十八方面，提供了一种重组多肽的制备方法，所述方法包括：
(a)在适合表达的条件下，培养如本申请第十四方面所述的宿主细胞；
(b)从培养物中分离出重组多肽，所述的重组多肽是如本申请第五方面所述的抗体或如本申请第六方面所述的重组蛋白。
本申请的第十九方面，提供了一种预防和/或治疗IL-36相关疾病的方法，所述方法包括：给需要的对象施用如本申请第五方面所述的抗体、所述抗体的抗体-药物偶联物、或表达所述抗体的CAR-T细胞、或其组合。
在某些实施方式中，所述的方法还包括：给需要的对象施用其他药物或治疗方法进行联合治疗。
应理解，在本申请范围内中，本申请的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的技术方案。限于篇幅，在此不再一一累述。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明书如下：
图1显示了人源化候选抗体生物活性检测-细胞因子IL-6释放结果。
图2显示了人源化候选抗体生物活性检测-细胞因子IL-8释放结果。
图3显示了人源化候选抗体生物活性检测-细胞因子GM-CSF释放结果。
图4显示了人源化候选抗体生物活性检测-NF-κB磷酸化结果。
图5显示了候选抗体和人IL-36R的细胞结合活性。
图6显示了候选抗体和猴IL-36R的细胞结合活性。
图7显示了候选抗体对huIL-36刺激因子在NCI/ADR-RES细胞的功能抑制作用。
图8显示了候选抗体对huIL-36刺激因子在HIF细胞的功能抑制作用。
图9显示了候选抗体对huIL-36刺激因子在HIF细胞的功能抑制作用。
图10显示了候选抗体对huIL-36刺激因子在HIF细胞的功能抑制作用。
图11显示了候选抗体对咪喹莫特诱导的银屑病食蟹猴临床评分(PASI)的影响。
图12显示了候选抗体对咪喹莫特诱导的银屑病食蟹猴皮肤组织HE染色病理评分的影响。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
本申请人经过广泛而深入地研究，意外地获得了高亲和力高生物活性的IL-36R抗体。实验表明，本申请的IL-36R抗体能够与IL-36R高亲和力结合，并且能够阻断IL-36R配体(α、β、γ)与IL-36R结合，阻断IL-36R配体激活的信号通路，从而治疗和/或预防IL-36相关疾病。在此基础上完成了本申请。
术语
如本文所用，术语“给予”和“处理”是指外源性药物、治疗剂、诊断剂或组合物应用于动物、人、受试者、细胞、组织、器官或生物流体。“给予”和“处理”可以指治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触、以及试剂与流体的接触、流体与细胞的接触。“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理。“处理”当应用于人、动物或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断；包括IL-36R抗体与人或动物、受试者、细胞、组织、生理区室或生理流体的接触。
如本文所用，术语“治疗”指给予患者内用或外用治疗剂，包含本申请的任何一种IL-36R 抗体及其组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，以有效缓解一种或多种疾病症状的治疗剂的量(治疗有效量)给予患者。
如本文所用，术语“任选”或“任选地”意味着随后所描述的事件或情况可以发生但不是必须发生。例如，“任选包含1-3个抗体重链可变区”是指特定序列的抗体重链可变区可以有但不是必须有，可以是1个、2个或3个。
抗体
如本文所用，术语“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的不同种型，即IgM、IgD、IgG、IgA和IgE，对应于不同类免疫球蛋白的重链恒定区分别称为α、δ、ε、γ和μ。IgG代表免疫球蛋白中最重要的一类，由于化学结构和生物功能差异，它又可以分为4个子类：IgG1、IgG2、IgG3和IgG4。轻链通过恒定区的不同分为κ或λ链。不同类免疫球蛋白的亚单位结构和三维构型是本领域人员所熟知的。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(V区)；靠近C端的其余氨基酸序列相对稳定，为恒定区(C区)。可变区包括3个高变区(HVR)和4个序列相对保守的FR区(FR)。4个FR的氨基酸序列相对比较保守，不直接参与结合反应。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(LCVR)和重链可变区(HCVR)由3个CDR区和4个FR区组成，从氨基端到羧基端依次排列的顺序为FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。轻链的3个CDR区，即轻链高变区(LCDR),指LCDR1、LCDR2和LCDR3；重链的3个CDR区，即重链高变区(HCDR),指HCDR1、HCDR2和HCDR3。发明所述的抗体或抗原结合片段的LCVR和HCVR区的CDR氨基酸残基在数量和位置符合已知的Kabat编号规则(LCDR1-3，HCDR2-3)，或者符合kabat和chothia的编号规则(HCDR1)。天然重链和轻链可变区中的四个FR区大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。
如本文所用，术语“抗原结合片段”，指具有抗原结合活性的Fab片段，Fab’片段，F(ab’)2片段，或单一Fv片段。Fv抗体含有抗体重链可变区、轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般的，Fv抗体还包含VH和VL结构域之间的多肽接 头，且能够形成抗原结合所需的结构。
如本文所用，术语“抗原决定簇”指抗原上不连续的，由本申请抗体或抗原结合片段识别的三维空间位点。
本申请不仅包括完整的抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此，本申请还包括所述抗体的片段、衍生物和类似物。
在本申请中，抗体包括用本领域技术人员熟知技术所制备的鼠的、嵌合的、人源化的或者全人的抗体。重组抗体，例如嵌合的和人源化的单克隆抗体，包括人的和非人的部分，可以采用本领域熟知的DNA重组技术制备。
如本文所用，术语“单克隆抗体”指得自单个细胞来源的克隆分泌的抗体。单克隆抗体是高度特异性的，针对单个抗原表位。所述的细胞可能是真核的、原核的或噬菌体的克隆细胞株。
如本文所用，术语“嵌合抗体”是由鼠源性抗体的V区基因与人抗体的C区基因拼接为嵌合基因，然后插入载体，转染宿主细胞表达的抗体分子。既保留了亲本鼠抗体的高特异性和亲和力，又使其人源Fc段能有效介导生物学效应功能。
如本文所用，术语“人源化抗体”，是本申请鼠抗的一种可变区改造形式，具有源自(或基本上源自)非人类抗体(例如可以为小鼠单克隆抗体)的CDR区，和基本源自人源抗体序列的FR区和恒定区；即将鼠抗的CDR区序列嫁接到不同类型的人种系抗体构架序列上。因为CDR序列负责大部分的抗体-抗原相互作用，所以可以通过构建表达载体来表达模拟特定天然存在的抗体性质的重组抗体。
在本申请中，抗体可以是单特异性、双特异性、三特异性、或者更多的多重特异性。
在本申请中，本申请的抗体还包括其保守性变异体，指与本申请抗体的氨基酸序列相比，有至多10个，可以为至多8个，可以为至多5个，可以为至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A
Figure PCTCN2021128704-appb-000001
Figure PCTCN2021128704-appb-000002
抗IL-36R抗体
如本文所用，术语“IL-36R”一般是指天然的或重组的人IL-36R，以及人IL-36R的非人同源物。除非另有指示，否则使用IL-36R的同源二聚体的分子量计算IL-36R的摩尔浓度。
本申请提供一种针对IL-36R的高特异性和高亲和力的抗体，其包括重链和轻链，所述重链含有重链可变区(VH)氨基酸序列，所述轻链含有轻链可变区(VL)氨基酸序列。
例如，重链可变区(VH)的CDR选自下组：
SEQ ID NO:3所示的CDR1，
SEQ ID NO:4所示的CDR2，和
SEQ ID NO:5所示的CDR3；和/或
轻链可变区(VL)的CDR选自下组：
SEQ ID NO:6所示的CDR1’，
SEQ ID NO:7所示的CDR2’，和
SEQ ID NO:8所示的CDR3’。
其中，上述氨基酸序列中任意一种氨基酸序列还包括经过添加、缺失、修饰和/或取代至少一个(如1-5、1-3个，可以为1-2个，可以为1个)氨基酸的具有IL-36R结合亲和力的衍生序列。
在某些实施方式中，所述经过添加、缺失、修饰和/或取代至少一个氨基酸序列所形成的序列同源性可以为至少80％，可以为至少85％，可以为至少为90％，可以为至少95％的氨基酸序列。
本申请的抗体可以是双链或单链抗体，并且可以是选自动物源抗体、嵌合抗体、人源化抗体，可以为人源化抗体、人-动物嵌合抗体，可以为全人源化抗体。
本申请所述抗体衍生物可以是单链抗体、和/或抗体片段，如：Fab、Fab’、(Fab’) 2或该领域内其他已知的抗体衍生物等，以及IgA、IgD、IgE、IgG以及IgM抗体或其他亚型的抗体中的任意一种或几种。
其中，所述动物可以为哺乳动物，如鼠。
本申请抗体可以是靶向人IL-36R的鼠源抗体、嵌合抗体、人源化抗体、CDR嫁接和/或修饰的抗体。
在某些情况下，上述SEQ ID NO：3、4和5中任意一种或几种序列、或它们经过添加、缺失、修饰和/或取代至少一个氨基酸的具有IL-36R结合亲和力的序列，位于重链可变区(VH)的CDR区。
在某些情况下，上述SEQ ID NO：6、7和8中任意一种或几种序列、或它们经过添加、缺失、修饰和/或取代至少一个氨基酸的具有IL-36R结合亲和力的序列，位于轻链可变区(VL)的CDR区。
在某些情况下，VH CDR1、CDR2、CDR3分别独立地选自SEQ ID NO：3、4和5中任意一种或几种序列、或它们经过添加、缺失、修饰和/或取代至少一个氨基酸的具有IL-36R结合亲和力的序列；VL CDR1、CDR2、CDR3分别独立地选自SEQ ID NO：6、7和8中任意一种或几种序列、或它们经过添加、缺失、修饰和/或取代至少一个氨基酸的具有IL-36R结合亲和力的序列。
本申请上述内容中，所述添加、缺失、修饰和/或取代的氨基酸数量，例如可以为不超过初始氨基酸序列总氨基酸数量的40％，可以为不超过35％，可以为1-33％，可以为5-30％，可以为10-25％，可以为15-20％。
在本申请中，所述添加、缺失、修饰和/或取代的氨基酸数量通常是1、2、3、4或5个，可以为1-3个，可以为1-2个，可以为1个。
抗体的制备
任何适于产生单克隆抗体的方法都可用于产生本申请的抗IL-36R抗体。例如，可以用连接或天然存在的IL-36R同源二聚体或其片段免疫动物。可以使用合适的免疫接种方法，包括佐剂、免疫刺激剂、重复加强免疫接种，可以使用一种或多种途径。
任何合适形式的IL-36R都可以作为免疫原(抗原)，用于产生对IL-36R特异的非人抗体，筛选所述抗体的生物学活性。激发免疫原可以是全长的成熟人IL-36R，包括天然的同源二聚体，或含单个/多个表位的肽。免疫原可以单独使用，或与本领域已知的一种或多种免疫原性增强剂组合使用。免疫原可以由天然来源纯化，或者在遗传修饰的细胞中产生。编码免疫原的DNA在来源上可以是基因组或非基因组的(例如cDNA)。可以使用合适的遗传载体表达编码免疫原的DNA，所述载体包括但不限于腺病毒载体、腺相关病毒载体、杆状病毒载体、质料和非病毒载体。
生产本申请的抗人IL-36R抗体的示例性方法描述于实施例1。
人源化抗体可以选自任何种类的免疫球蛋白，包括IgM、IgD、IgG、IgA和IgE。在本申请中，抗体是IgG抗体，使用IgG1亚型。通过用下文实施例中描述的生物学测定筛选抗体易于实现必需恒定结构域序列的最优化，以产生所需生物学活性。
同样，任一类轻链都可以在本文的化合物和方法中使用。具体地说，κ、λ链或其变体在本申请的化合物和方法中是可以用的。
人源化本申请的抗人IL-36R抗体的示例性方法描述于实施例2。
本申请抗体或其片段的DNA分子的序列可以用常规技术，比如利用PCR扩增或基因组文库筛选等方法获得。此外，还可将轻链和重链的编码序列融合在一起，形成单链抗体。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。
本申请还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。动物细胞可以包括(但并不限于)：CHO-S、CHO-K1、HEK-293细胞。
本申请中所述的用重组DNA转化宿主细胞的步骤可用本领域熟知的技术进行。获得的转化子可用常规方法培养，转化子表达本申请的基因所编码的多肽。根据所用的宿主细胞，用常规培养基在合适的条件下培养。
通常，在适合本申请抗体表达的条件下，培养转化所得的宿主细胞。然后用常规的免疫球蛋白纯化步骤，如蛋白A-Sepharose、羟基磷灰石层析、凝胶电泳、透析、离子交换层析、疏水层析、分子筛层析或亲和层析等本领域技术人员熟知的常规分离纯化手段纯化得到本申请的抗体。
所得单克隆抗体可用常规手段来鉴定。比如，单克隆抗体的结合特异性可用免疫沉淀或体外结合试验(如放射性免疫测定(RIA)或酶联免疫吸附测定(ELISA))来测定。
抗体-药物偶联物(ADC)
本申请还提供了基于本申请抗体的抗体偶联药物(antibody-drug conjugate，ADC)。
典型地，所述抗体偶联药物包括所述抗体、以及效应分子，所述抗体与所述效应分子偶联，并可以为化学偶联。其中，所述效应分子可以为具有治疗活性的药物。此外，所述效应分子可以是毒蛋白、化疗药物、小分子药物或放射性核素中的一种或多种。
本申请抗体与所述效应分子之间可以是通过偶联剂进行偶联。所述偶联剂的例子可以是非选择性偶联剂、利用羧基的偶联剂、肽链、利用二硫键的偶联剂中的任意一种或几种。所述非选择性偶联剂是指使效应分子和抗体形成共价键连接的化合物，如戊二醛等。所述利用羧基的偶联剂可以是顺乌头酸酐类偶联剂(如顺乌头酸酐)、酰基腙类偶联剂(偶联位点为酰基腙)中的任意一种或几种。
抗体上某些残基(如Cys或Lys等)用于与多种功能基团相连，其中包括成像试剂(例如发色基团和荧光基团)，诊断试剂(例如MRI对比剂和放射性同位素)，稳定剂(例如乙二醇聚合物)和治疗剂。抗体可以被偶联到功能剂以形成抗体-功能剂的偶联物。功能剂(例如药物，检测试剂，稳定剂)被偶联(共价连接)至抗体上。功能剂可以直接地、或者是通过接头间接地连接于抗体。
抗体可以偶联药物从而形成抗体药物偶联物(ADCs)。典型地，ADC包含位于药物和抗体之间的接头。接头可以是可降解的或者是不可降解的接头。可降解的接头典型地在细胞内环境下容易降解，例如在目标位点处接头发生降解，从而使药物从抗体上释放出来。合适的可降解的接头包括，例如酶降解的接头，其中包括可以被细胞内蛋白酶(例如溶酶体蛋白酶或者内体蛋白酶)降解的含有肽基的接头，或者糖接头例如，可以被葡糖苷酸酶降解的含葡糖苷酸的接头。肽基接头可以包括，例如二肽，例如缬氨酸-瓜氨酸，苯丙氨酸-赖氨酸或者缬氨酸-丙氨酸。其它合适的可降解的接头包括，例如，pH敏感接头(例如pH小于5.5时水解的接头，例如腙接头)和在还原条件下会降解的接头(例如二硫键接头)。不可降解的接头典型地在抗体被蛋白酶水解的条件下释放药物。
连接到抗体之前，接头具有能够和某些氨基酸残基反应的活性反应基团，连接通过活性反应基团实现。巯基特异性的活性反应基团是可以包括：例如马来酰亚胺类化合物，卤代酰 胺(例如碘、溴或氯代的)；卤代酯(例如碘、溴或氯代的)；卤代甲基酮(例如碘、溴或氯代)，苄基卤代物(例如碘、溴或氯代的)；乙烯基砜，吡啶基二硫化物；汞衍生物例如3,6-二-(汞甲基)二氧六环，而对离子是醋酸根、氯离子或者硝酸根；和聚亚甲基二甲基硫醚硫代磺酸盐。接头可以包括，例如，通过硫代丁二酰亚胺连接到抗体上的马来酰亚胺。
药物可以是任何细胞毒性，抑制细胞生长或者免疫抑制的药物。在实施方式中，接头连接抗体和药物，而药物具有可以和接头成键的功能性基团。例如，药物可以具有可以和连接物成键的氨基，羧基，巯基，羟基，或者酮基。在药物直接连接到接头的情况下，药物在连接到抗体之前，具有反应的活性基团。
有用的药物类别包括，例如，抗微管蛋白药物、DNA小沟结合试剂、DNA复制抑制剂、烷化试剂、抗生素、叶酸拮抗物、抗代谢药物、化疗增敏剂、拓扑异构酶抑制剂、长春花生物碱等。在本申请中，药物-接头可以用于在一个简单步骤中形成ADC。在其它实施方式中，双功能连接物化合物可以用于在两步或多步方法中形成ADC。例如，半胱氨酸残基在第一步骤中与接头的反应活性部分反应，并且在随后的步骤中，接头上的功能性基团与药物反应，从而形成ADC。
通常，选择接头上功能性基团，以利于特异性地与药物部分上的合适的反应活性基团进行反应。作为非限制性的例子，基于叠氮化合物的部分可以用于特异性地与药物部分上的反应性炔基基团反应。药物通过叠氮和炔基之间的1,3-偶极环加成，从而共价结合于接头。其它的有用的功能性基团包括，例如酮类和醛类(适合与酰肼类和烷氧基胺反应)，膦(适合与叠氮反应)；异氰酸酯和异硫氰酸酯(适合与胺类和醇类反应)；和活化的酯类，例如N-羟基琥珀酰亚胺酯(适合与胺类和醇类反应)。这些和其它的连接策略，例如在《生物偶联技术》，第二版(Elsevier)中所描述的，是本领域技术人员所熟知的。本领域技术人员能够理解，对于药物部分和接头的选择性反应，当选择了一个互补对的反应活性功能基团时，该互补对的每一个成员既可以用于接头，也可以用于药物。
本申请还提供了制备ADC的方法，可进一步地包括：将抗体与药物-接头化合物，在足以形成抗体偶联物(ADC)的条件下进行结合。
在某些实施方式中，本申请方法包括：在足以形成抗体-接头偶联物的条件下，将抗体与双功能接头化合物进行结合。在这些实施方式中，本申请方法还进一步地包括：在足以将药物部分通过接头共价连接到抗体的条件下，将抗体接头偶联物与药物部分进行结合。
在一些实施方式中，抗体药物偶联物ADC如下分子式所示：
Figure PCTCN2021128704-appb-000003
其中：
Ab是抗体，
LU是接头；
D是药物；
而且下标p是选自1到8的值。
应用
本申请提供了本申请抗体的用途，例如用于制备诊断制剂、或制备用于预防和/或治疗IL-36相关疾病的药物。所述IL-36相关疾病包括炎症疾病、自身免疫疾病等，包括但不限于银屑病、银屑病关节炎、强直性脊柱炎、多发性硬化症，炎性肠病(如克罗恩氏病、溃疡性结肠炎等)、骨关节炎、类风湿性关节炎(RA)、风湿性关节炎或骨质疏松症、炎性纤维化(例如硬皮病、肺纤维化和硬化)、哮喘(包括变应性哮喘)、变态反应以及癌症。
药物组合物
本申请还提供了一种组合物。在某些情况下，所述的组合物是药物组合物，它含有上述的抗体或其活性片段或其融合蛋白或其ADC或相应的CAR-T细胞，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，可以为pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：瘤内、腹膜内、静脉内、或局部给药。
本申请所述抗体也可以是由核苷酸序列在细胞内表达用于的细胞治疗，比如，所述抗体用于嵌合抗原受体T细胞免疫疗法(CAR-T)等。
本申请的药物组合物可直接用于结合IL-36R蛋白分子，因而可用于预防和治疗IL-36相关疾病。此外，还可同时使用其他治疗剂。
本申请的药物组合物含有安全有效量(如0.001-99wt％,可以为0.01-90wt％，可以为0.1-80wt％)的本申请上述的单克隆抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本申请的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约1微克/千克体重-约5毫克/千克体重。此外，本申请的多肽还可与其他治疗剂一起使用。
使用药物组合物时，是将安全有效量的药物组合物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，可以为该剂量是约10微克/千克体重-约20毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健 康状况等因素，这些都是熟练医师技能范围之内的。
检测用途和试剂盒
本申请的抗体可用于检测应用，例如用于检测样本，从而提供诊断信息。
本申请中，所采用的样本(样品)包括细胞、组织样本和活检标本。本申请使用的术语“活检”应包括本领域技术人员已知的所有种类的活检。因此本申请中使用的活检可以包括例如通过内窥镜方法或器官的穿刺或针刺活检制备的组织样本。
本申请中使用的样本包括固定的或保存的细胞或组织样本。
本申请还提供了一种指含有本申请的抗体(或其片段)的试剂盒，例如，所述的试剂盒还可以包括容器、使用说明书、缓冲剂等。例如，本申请的抗体可以固定于检测板。
本申请涉及的抗体或其片段可以具有以下的优异技术效果：
(a)本申请抗体具有优异的生物活性和特异性。
(b)与鼠源抗体和嵌合抗体相比，本申请人源化抗体在保留与IL-36R相当的亲合力的同时，具有更低的免疫原性。
(c)本申请抗体对IL-36R的阻断可以显著抑制患者炎症因子表达通路。
(d)本申请抗体与某些非人哺乳动物(如猴)的IL-36R有与人IL-36R相当的亲和力，便于在动物模型中进行测试和进行质控检测。
(e)本申请抗体通过与IL-36R结合阻断IL-36α、IL-36β、IL-36γ与IL-36R结合的激活信号通路，减少炎症细胞因子(如IL-6、IL-8、GM-CSF)
下面结合具体实施例，进一步阐述本申请。应理解，这些实施例仅用于说明本申请而不用于限制本申请的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例
实施例1
制备鼠源单克隆抗体的方法采用Kohler和Milstein 1975年发明的杂交瘤制备技术(Nature，1975，256：495-497)。首先将人IL-36R带有Fc标签蛋白(ACRO，#IL2-H5254)与弗氏佐剂乳化，然后对多个品系小鼠进行免疫。四轮免疫后取血清用ELISA法检测效价，挑选最佳小鼠取脾细胞与SP2/0骨髓瘤细胞进行融合。经过HAT筛选杂交瘤多克隆细胞，对多克隆培养上清进行人IL-36R、猴IL-36R、人IL1R1的结合活性检测，以及多克隆抗体对IL- 36刺激因子的功能抑制作用检测。挑选最佳多克隆进行单克隆化，同样对单克隆进行结合活性以及功能活性的筛选，并通过Biacore方法检测亲和力，最终将挑选出的最佳杂交瘤单克隆细胞株进行序列分析。
表1.杂交瘤筛选部分数据
Figure PCTCN2021128704-appb-000004
如表1所示，经过大量杂交瘤筛选，杂交瘤6(1)-B8，111(1)-E5抗体表达上清在细胞结合水平与人IL-36R、猴IL-36R蛋白均有很高的结合活性，与人IL1R1蛋白无特异性结 合，并对IL-36刺激因子(IL-36α，IL-36β，IL-36γ)的刺激产生的细胞因子有明显的功能阻断作用。
实施例2抗IL-36R抗体可变区基因序列克隆以及人源化
2.1杂交瘤细胞中抗体的可变区基因克隆
基于TAKARA的5’RACE技术原理，克隆出由杂交瘤细胞株表达的小鼠抗体可变区的cDNA序列。简言之，用SMARTer 5’RACE合成试剂盒(TAKARA，货号634859)按说明书合成重链和轻链的可变区基因特异性cDNA。用PCR引物修饰cDNA序列的5’和3’端，所述引物设计成为分别在重链和轻链可变区cDNA上增加合适的前导序列，使所得PCR产物能够通过无缝克隆的方法克隆到现有重组抗体表达的重链载体pHB-Fc和轻链载体pHB-Cκ上。pHB-Fc表达载体上含有人IgG1重链恒定区基因序列，其中CH2上带有抗体ADCC效应弱化的L234A和L235A(Eu numbering)突变；pHB-Cκ载体上含有人κ轻链恒定区基因序列。将重链和轻链可变区PCR扩增产物通过In-fusion克隆试剂(TAKARA,货号639650)克隆到表达载体上，获得人鼠嵌合抗体表达载体，并转化到E.coli DH5α大肠杆菌感受态细胞(益生生技，货号FYE607-80VL)中。通过挑选单克隆菌落进行Sanger测序，经分析获得抗体可变区序列。
结果获得抗IL-36R嵌合抗体(华博编号900497)，其轻链来源于实施例1中的杂交瘤6(1)-B8，重链来源于实施例1中的杂交瘤111(1)-E5，其可变区序列如下所示：
900497 HCVR SEQ ID NO：1
Figure PCTCN2021128704-appb-000005
900497 LCVR SEQ ID NO：2
Figure PCTCN2021128704-appb-000006
其中，下划线区为CDRs(IMGT定义，单列如下)：
表2鼠源抗IL-36R抗体CDR序列
Figure PCTCN2021128704-appb-000007
2.2嵌合抗体的表达
2.1中所得表达载体经过大肠杆菌扩增，用去内毒素质粒抽提试剂盒(天根生化科技(北京)有限公司，货号DP117)制备足量质粒，用于瞬时转染表达嵌合抗体。表达所用的宿主细胞为CHO-S细胞(赛默飞，货号R80007)。通过将制备所得的两种重链载体分别和轻链载体一起，与聚醚酰亚胺(PEI，Polysciences，货号24765-1)混合形成脂质体复合物后，转染CHO-S细胞，放入二氧化碳摇床中培养5-7天。离心收集细胞培养液上清，通过Protein A亲和层析柱纯化得到人鼠嵌合抗体。
2.3鼠源抗人IL-36R抗体的人源化——人源化抗体的制备方法
抗体的人源化采用以下方法：将嵌合抗体(华博编号900497)的可变区序列与NCBI IgBlast数据库中的可用序列比较，通过鉴定和分析，最终确定了适合在其上构建CDR移植重链和轻链的人源构架区(FR区)。
改造时，根据人抗体FR区保守的氨基酸残基以及抗体FR区中重要的氨基酸残基，设计改造位点，对嵌合抗体的重轻链的可变区分别进行人源化突变设计，利用PCR技术扩增并构建人源化点突变抗体表达质粒。将人源化点突变抗体表达质粒分别经CHO-S细胞表达，纯化后得到人源化抗体蛋白。利用Biacore和细胞生物活性等检测方法，对人源化抗体的亲和力，激活细胞因子释放等指标进行筛选，获得了三个性能优异的人源化抗IL-36R抗体。所获得人源化抗IL-36R抗体的编号为900513、900527和900534，其VH和VL序列下所示：
900513、900527和900534 HCVR SEQ ID NO：9
Figure PCTCN2021128704-appb-000008
900513 LCVR SEQ ID NO：10
Figure PCTCN2021128704-appb-000009
900527 LCVR SEQ ID NO：11
Figure PCTCN2021128704-appb-000010
900534 LCVR SEQ ID NO：12
Figure PCTCN2021128704-appb-000011
其中，下划线区为CDRs(IMGT定义)，900513，900527和900534分别为三个人源化抗体蛋白编号。人源化抗体900513，900527和900534的CDR区与嵌合抗体900497的CDR区 相同,人源化抗体的FR区在嵌合900497的基础上进行了突变。
实施例3抗人IL-36R人源化抗体生物活性检测
3.1 Biacore(SPR)检测抗体亲和力
试验材料与仪器如下：
His标记的人IL-1 Rrp2/IL-1 R6蛋白(Human IL-1 Rrp2/IL-1 R6 Protein,His Tag)，ACRO Biosystems，IL2-H52H6
HBS-EP+(10X)，GE，BR-1006-69
Series S Sensor Chip CM5，GE，29-1275-56
His Capture Kit，GE，28995056
BIACORE，GE，Biacore 8K
pH1.5甘氨酸溶液，GE,BR100354
试验方法如下：
Sereis S Sensor Chip CM5芯片室温平衡20-30min，将芯片装入仪器。按照His Capture Kit说明书固定anti-His抗体到Series S Sensor Chip CM5芯片上。HBS-EP+(10X)溶液超纯水稀释10倍作为运行缓冲液(Running Buffer)。利用运行缓冲液将抗原(His标记的人IL-1 Rrp2/IL-1 R6蛋白)稀释至1μg/ml，10μL/min，注入15秒，捕获抗原50RU左右。利用平衡缓冲液将抗体样品稀释至30nM，15nM，7.5nM，3.75nM，1.875nM，0.9375nM，0.46875nM；运行缓冲液作为零浓度；30μL/min，结合120s，解离900s；pH1.5甘氨酸溶液再生芯片。采用捕获法单循环动力学程序分析样品，选用对应的分析程序对数据分析，确认无明显参考结合(reference binding)，选用Kinetics，1:1结合型(binding modle)，拟合分析，获得样品的动力学参数。
表3候选抗人IL-36R人源化抗体的亲和力测定
蛋白编号 ka(1/Ms) kd(1/s) KD(M)
900513 9.04E+05 1.34E-05 1.48E-11
900527 9.67E+05 4.37E-05 4.52E-11
900534 8.81E+05 5.10E-05 5.79E-11
900497 6.60E+05 1.68E-05 2.54E-11
900389 7.39E+05 1.17E-05 1.58E-11
注：900389为BI研发的抗人IL-36R抗体，由华博根据专利(WO2013/074569A1)中记 载的序列合成可变区基因，自主表达而得。具体地，900389的重链可变区为WO2013/074569A1中SEQ ID NO:89所示的81B4vH33_90vH，900389的轻链可变区为WO2013/074569A1中SEQ ID NO:77所示的81B4vK32_105vK，81B4vH33_90vH和81B4vK32_105vK均是在鼠源抗体81B4基础上进行人源化改造获得的；900389的恒定区与本申请候选抗体相同。
各人源化抗体与人IL-1 Rrp2结合的亲和力常数(KD(M))如表3所示。结果显示，本申请人源化单克隆抗体的亲和力接近10 -11数量级，具有极强的亲和力。
3.2抗人IL-36R抗体对huIL-36刺激因子的功能抑制作用检测
将人卵巢癌细胞系NCI/ADR-RES以每孔100μL(每孔细胞数4.5×10 4个)加入96孔细胞培养板，37℃，5％CO 2孵育过夜，待测样品系列稀释后50μL/孔加入，37℃，5％CO 2孵育15min，分别将配体huIL-36α(1μg/mL)、huIL-36β(80ng/mL)、huIL-36γ(120ng/mL)以50μL/孔加入，充分混匀后，37℃，5％CO 2培养18-24h。离心取细胞培养上清，用Biolegend ELISA试剂盒检测：huIL-6(1:100稀释，货号430503)、huIL-8(1:5稀释，货号431503)和huGM-CSF(1:2稀释，货号432003)。检测huIL-36β刺激作用下，NCI/ADR-RES细胞内NF-κB的激活作用：将配体huIL-36β稀释至240ng/mL，加入与待测抗体预孵过的细胞中，37℃，5％CO 2孵育30min。离心取出上清，将细胞裂解后取裂解液用试剂盒(Cisbio，货号64NFBPEG)进行NF-κB磷酸化检测。
表4人源化候选抗体生物活性检测结果
Figure PCTCN2021128704-appb-000012
结果如图1-图4和表4所示，人源化抗体900513,900527,900534对人的刺激因子IL-36α，IL-36β，IL-36γ的功能具有显著的阻断作用，能够显著阻断细胞因子IL-6、IL-8、GM-CSF的 分泌和NF-κB的磷酸化。
实施例4嵌合抗体和人源化抗体的非特异性结合实验(SPR)
本试验使用SPR方法测定抗体与非靶标分子的非特异吸附效应，试验中使用的仪器设备和试剂材料如表5和表6所示。
表5仪器设备
名称 厂家 型号 编号
Biacore GE Biacore 8K 1305
表6试剂材料：
Figure PCTCN2021128704-appb-000013
将Series S Sensor Chip CM5(GE，#BR-1005-30)芯片置于室温平衡20～30min，装入Biacore 8K(GE)仪器。采用氨基偶联试剂盒(GE，#BR-1000-50)将来自鸡蛋的溶菌酶溶液(Sigma，#L3790)和来自大豆的胰蛋白酶抑制剂1-S型(Sigma，#T-2327)分别固定到CM5芯片。进样缓冲液为HBS-EP(1X)(GE，#BR-1006-69)，设置4个平衡循环。用平衡缓冲液分别将多克隆兔抗溶菌酶(ABcam，Ab391)，抗胰蛋白酶抑制剂抗体(Anti-trypsin inhibitor antibody,LifeSpan Biosciences，#LS-C76609)，嵌合抗体和人源化抗体稀释至1000nM，设置流速5μL/min，进样通道1，2和3，Flow Cell 1和2。结合时间10min，解离时间15min。再生流速50μL/min，先用0.85％磷酸溶液(ProteOn，176-2260)再生60s，再用50mM氢氧化钠溶液再生30s。
其中，样品900389为BI研发的抗人IL-36R人源化抗体，由华博根据专利合成可变区基因，自主表达而得。样品900497为华博杂交瘤筛选构建的抗IL-36R嵌合抗体。
结果表明如表7所示，900389与来自鸡蛋的溶菌酶溶液的结合信号大于20RU，因而认为有非特异性的静电和疏水结合作用。900497与来自鸡蛋的溶菌酶溶液和来自大豆的胰蛋白 酶抑制剂1-S型的结合信号均小于20，因此可以认为无非特异性的静电和疏水结合作用。
表7样品非特异性结合量比较
Figure PCTCN2021128704-appb-000014
注：一般认为扣除Buffer的影响后，响应值在20RU以下相互作用较弱，可以忽略；超过20RU即认为有明显的相互作用；100RU以上则是很强的相互作用。
实施例5稳定细胞株抗体的细胞结合和功能实验
将BI对照序列900389，华博筛选的人源化序列900527,TNP IgG1(Fc silence)900543分别插入华博GS载体，构建重组表达质粒并分别转染CHO-K1细胞进行稳定细胞株的构建。华博900527构建的稳定细胞株蛋白编号为HB0034，筛选的单克隆细胞株经过上游工艺优化后表达量超过4g/L。将稳转细胞株表达的抗体纯化后进行流式细胞结合实验和功能实验检测。样品900543为TNP IgG1(Fc silence)阴性对照抗体。
5.1稳定细胞株抗体的流式细胞结合实验
将稳定细胞株抗体进行人IL-36R、猴IL-36R的结合活性检测，流式检测细胞株为华博内部构建的表达人IL-36R、猴IL-36R的CHO-K1细胞株。
检测结果如表8和图5、图6所示。
表8抗体和人IL-36R、猴IL-36R的结合活性检测
Figure PCTCN2021128704-appb-000015
结果显示，华博单克隆抗体HB0034和人IL-36R,猴IL-36R均有很高的结合活性。相较于阳性对照900389抗体，本申请抗体的EC50值更低，具有与人IL-36R更强的结合活性。
5.2稳定细胞株抗体对huIL-36刺激因子的功能抑制作用检测实验
5.2.1抗体对huIL-36刺激因子在NCI/ADR-RES细胞的功能抑制作用
将人卵巢癌细胞系NCI/ADR-RES以每孔100μL(每孔细胞数4.5×10 4个)加入96孔细胞培养板，37℃，5％CO 2孵育过夜，待测样品系列稀释后50μL/孔加入，37℃，5％CO 2孵育15min，将配体huIL-36β(40ng/mL)以50μL/孔加入，充分混匀后，37℃，5％CO 2培养18-24h。离心取细胞培养上清，用HTRF试剂盒(Cat#62HIL06PEG，CISBIO)检测huIL-6。
结果如表9和图7所示。
表9抗体对huIL-36刺激因子在NCI/ADR-RES细胞的功能抑制作用
Figure PCTCN2021128704-appb-000016
结果显示，稳定细胞株人源化抗体HB0034对人的刺激因子IL-36β的功能具有显著的阻断作用，能够显著阻断细胞因子IL-6的分泌。相较于阳性对照900389抗体，本申请抗体的IC50值更低，具有更强的阻断作用。
5.2.2抗体对huIL-36刺激因子在HIF细胞的功能抑制作用
将HIF(Human Intestinal Fibroblasts)细胞用Serum Starvd培养基将细胞制备成4.5E5/ml细胞悬液，每孔100ul(45000个细胞)加入96wp,孵育过夜。抗体系列稀释后加入50ul/孔，37℃孵育15min。IL-36α稀至1000ng/ml,每孔加50ul，充分混合。37℃，5％CO 2培养4h，离心收集上清，检测人IL-6的含量。IL-36β稀至80ng/m,每孔加50ul，充分混合。37℃，5％CO 2培养4h，离心收集上清，检测人IL-6的含量。IL-36β稀至40ng/m,每孔加50ul，充分混合。37℃，5％CO 2培养20min，利用试剂盒检测pNFκB的含量。
结果如表10和图8，表11和图9，表12和图10。
表10抗体对huIL-36刺激因子在HIF细胞的功能抑制作用
Figure PCTCN2021128704-appb-000017
表11抗体对huIL-36刺激因子在HIF细胞的功能抑制作用
Figure PCTCN2021128704-appb-000018
表12抗体对huIL-36刺激因子在HIF细胞的功能抑制作用
Figure PCTCN2021128704-appb-000019
结果显示，稳定细胞株人源化抗体HB0034对人的刺激因子IL-36α，IL-36β的功能具有显著的阻断作用，能够显著阻断细胞因子IL-6的分泌和NF-κB的磷酸化。相较于阳性对照900389抗体，本申请抗体的IC50值更低，具有更强的阻断作用。
实施例6人源化抗体的非特异性结合实验(SPR)
本试验使用SPR方法测定抗体样品(900389和HB0034)与非靶标分子的非特异吸附效应，试验中使用的试剂材料如表13所示。
表13试剂材料
Figure PCTCN2021128704-appb-000020
将Series S Sensor Chip CM5(GE，#BR-1005-30)芯片置室温平衡20～30min，装入Biacore 8K(GE)仪器。使用氨基偶联试剂盒(GE，#BR-1000-50)将来自鸡蛋的溶菌酶溶液(Sigma，#L3790)和来自大豆的胰蛋白酶抑制剂1-S型(Sigma，#T-2327)分别固定到CM5芯片通 道1和2。运行缓冲液为HBS-EP+(1X)(GE，#BR-1006-69)，设置5个平衡循环。用运行缓冲液将多克隆兔抗溶菌酶(ABcam，#Ab391)，Anti-trypsin inhibitor antibody(LifeSpan Biosciences，#LS-C76609)，嵌合抗体和人源化抗体稀释至1000nM，设置流速5μL/min，进样通道1，2和3，Flow Cell 1和2。结合时间10min，解离时间15min。再生流速50μL/min，先0.85％磷酸溶液(BIO-RAD，#176-2260)再生60s，再用50mM氢氧化钠溶液(GE,#BR-1003-58)再生30s。
结果表明如表14所示，BI对照抗体900389与溶菌酶的结合响应值大于20RU，而与胰蛋白酶的结合响应值小于20RU，表明该样品带有负电荷。而在本次实验中，900389与羧甲基葡聚糖有53.9RU的结合响应值，表明该样品可结合羧甲基葡聚糖。HB0034与溶菌酶和胰蛋白酶的结合响应值值均小于20RU，可认为该样品没有明显非特异性的静电结合作用。
表14样品非特异性结合量比较
Figure PCTCN2021128704-appb-000021
注：一般认为扣除Buffer的影响后，响应值在20RU以下相互作用较弱，可以忽略；超过20RU即认为有明显的相互作用；100RU以上则是很强的相互作用。
实施例7人源化抗体对银屑病的治疗效果
将食蟹猴分为5组，每组6只，分别为模型对照组、阳性对照药组、HB0034低剂量组、HB0034中剂量组及HB0034高剂量组。咪喹莫特乳膏(IMQ)涂抹，形成银屑病模型。首次造模当天(Day0)开始及第4天(Day4)，HB0034低、中、高剂量组分别静脉注射给予5、15、50mg/kg HB0034，模型对照组给予生理盐水，阳性对照药组给予地塞米松(5mg/cm 2，涂抹给药，每天一次)。
实验结果显示，试验结束时，各组体重均略有下降，HB0034治疗组随着剂量的增加，体重下降幅度有减少趋势；与生理盐水(模型组)相比，在5mg/kg剂量下，HB0034显著降低造模皮肤组织中IL-17、IL-36的水平(p＜0.05)，对皮肤病理评分有改善趋势，但差异不显著 (p＞0.05)(见表15)；在15mg/kg剂量下，HB0034对模型PASI评分有明显改善(实验终点Day10，p＜0.05)，对皮肤病理评分有改善趋势，但差异不显著(p＞0.05)(见表15，图12)；对造模皮肤组织中IL-17、IL-36的水平有显著降低(p＜0.05)；在50mg/kg剂量下，HB0034对模型PASI评分有显著改善(Day4～10，p<0.05)，对皮肤病理评分和皮肤IL-17、IL-36的水平均有显著降低(p＜0.05)(见表15，图11-图12)。其中，图11中，n＝6,*p<0.05,**p<0.01vs.生理盐水；图12中，n＝6,**p<0.01vs.生理盐水。
阳性对照药地塞米松(5mg/cm 2)在该模型中的药效略优于15mg/kg HB0034，稍弱于50mg/kg HB0034。
由此可见，HB0034在15～50mg/kg剂量下(i.v.，Q4D×2)，剂量依赖性地改善IMQ诱导的皮肤红斑、皮屑及皮肤增厚的银屑病样症状，降低PASI评分，改善造模部位皮肤病理评分和降低皮肤组织IL-17、IL-36水平。HB0034在IMQ诱导的食蟹猴银屑病模型中，最小起效剂量为15mg/kg。
表15 HB0034对咪喹莫特诱导的食蟹猴银屑病模型皮肤组织IL-17及IL-36水平的影响
Figure PCTCN2021128704-appb-000022
其中，mean±SEM,n＝6；*p<0.05,**p<0.01vs.生理盐水
在本申请提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本申请的上述讲授内容之后，本领域技术人员可以对本申请作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (17)

  1. 一种抗体，其特征在于，所述抗体具有：
    (1)重链可变区，所述的重链可变区包括以下三个互补决定区CDR：
    SEQ ID NO:3所示的CDR1，
    SEQ ID NO:4所示的CDR2，和
    SEQ ID NO:5所示的CDR3；和
    (2)轻链可变区，所述的轻链可变区包括以下三个互补决定区CDR：
    SEQ ID NO:6所示的CDR1’，
    SEQ ID NO:7所示的CDR2’，和
    SEQ ID NO:8所示的CDR3’。
  2. 如权利要求1所述的抗体，其特征在于，所述抗体选自：动物源抗体、嵌合抗体、人源化抗体、或其组合。
  3. 如权利要求1所述的抗体，其特征在于，所述抗体的重链可变区序列如SEQ ID NO：1或9所示；和/或
    所述抗体的轻链可变区序列如SEQ ID NO：2、10、11或12所示。
  4. 一种重组蛋白，其特征在于，所述的重组蛋白具有：
    (i)如权利要求1所述的抗体；以及
    (ii)任选的协助表达和/或纯化的标签序列。
  5. 一种CAR构建物，其特征在于，所述的CAR构建物的单克隆抗体抗原结合区域的scFv段为特异性结合于IL-36R的结合区，
    并且，所述scFv的重链可变区包括以下三个互补决定区CDR：
    SEQ ID NO:3所示的CDR1，
    SEQ ID NO:4所示的CDR2，和
    SEQ ID NO:5所示的CDR3；和
    所述scFv的轻链可变区包括以下三个互补决定区CDR：
    SEQ ID NO:6所示的CDR1’，
    SEQ ID NO:7所示的CDR2’，和
    SEQ ID NO:8所示的CDR3’。
  6. 一种重组的免疫细胞，其特征在于，所述的免疫细胞表达外源的如权利要求5所述的CAR构建物。
  7. 一种抗体药物偶联物，其特征在于，所述的抗体药物偶联物含有：
    (a)抗体部分，所述抗体部分为如权利要求1所述的抗体；和
    (b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、或其组合。
  8. 一种活性成分的用途，所述活性成分选自下组：如权利要求1所述的抗体、如权利要求4所述的重组蛋白、如权利要求5所述的CAR构建物、如权利要求6所述的免疫细胞、如权利要求7所述的抗体药物偶联物、或其组合，其特征在于，所述活性成分用于(a)制备检测试剂或试剂盒；和/或(b)制备预防和/或治疗IL-36相关疾病的药物。
  9. 如权利要求8所述用途，其特征在于，所述的IL-36相关疾病包括银屑病。
  10. 一种药物组合物，其特征在于，所述的药物组合物含有：
    (i)活性成分，所述活性成分选自下组：如权利要求1所述的抗体、如权利要求4所述的重组蛋白、如权利要求5所述的CAR构建物、如权利要求6所述的免疫细胞、如权利要求7所述的抗体药物偶联物、或其组合；以及
    (ii)药学上可接受的载体。
  11. 一种多核苷酸，其特征在于，所述的多核苷酸编码如权利要求1所述的抗体、如权利要求4所述的重组蛋白、或如权利要求5所述的CAR构建物。
  12. 一种载体，其特征在于，所述的载体含有如权利要求11所述的多核苷酸。
  13. 一种遗传工程化的宿主细胞，其特征在于，所述的宿主细胞含有如权利要求12所述的载体或基因组中整合有如权利要求11所述的多核苷酸。
  14. 一种非诊断性体外检测样品中IL-36R蛋白的方法，其特征在于，所述方法包括步骤：
    (1)在体外，将所述样品与如权利要求1所述的抗体或如权利要求7所述的抗体药物偶联物、接触；
    (2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在IL-36R蛋白。
  15. 一种检测板，其特征在于，所述的检测板包括：基片(支撑板)和测试条，所述的测试条含有如权利要求1所述的抗体或如权利要求7所述的抗体药物偶联物。
  16. 一种试剂盒，其特征在于，所述试剂盒中包括：
    (1)第一容器，所述第一容器中含有如权利要求1所述的抗体；和/或
    (2)第二容器，所述第二容器中含有针对如权利要求1所述的抗体的二抗；
    或者，所述试剂盒含有如权利要求15所述的检测板。
  17. 一种重组多肽的制备方法，其特征在于，所述方法包括：
    (a)在适合表达的条件下，培养如权利要求13所述的宿主细胞；
    (b)从培养物中分离出重组多肽，所述的重组多肽是如权利要求1所述的抗体或如权利要求4所述的重组蛋白。
PCT/CN2021/128704 2020-11-04 2021-11-04 靶向于白介素36r的抗体及其制备方法和应用 WO2022095926A1 (zh)

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KR1020237017515A KR20230098606A (ko) 2020-11-04 2021-11-04 인터루킨 36r을 표적으로 하는 항체 및 이의 제조 방법과 용도
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