WO2022166977A1 - 抗人il-36r抗体及其应用 - Google Patents

抗人il-36r抗体及其应用 Download PDF

Info

Publication number
WO2022166977A1
WO2022166977A1 PCT/CN2022/075460 CN2022075460W WO2022166977A1 WO 2022166977 A1 WO2022166977 A1 WO 2022166977A1 CN 2022075460 W CN2022075460 W CN 2022075460W WO 2022166977 A1 WO2022166977 A1 WO 2022166977A1
Authority
WO
WIPO (PCT)
Prior art keywords
human
antibody
amino acid
fragment
acid sequence
Prior art date
Application number
PCT/CN2022/075460
Other languages
English (en)
French (fr)
Inventor
王晋
邓小芳
李祥烽
徐晓红
高攀
Original Assignee
上海普铭生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 上海普铭生物科技有限公司 filed Critical 上海普铭生物科技有限公司
Publication of WO2022166977A1 publication Critical patent/WO2022166977A1/zh

Links

Images

Classifications

    • 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
    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N5/0684—Cells of the urinary tract or kidneys
    • C12N5/0686—Kidney cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • C12N5/163—Animal cells one of the fusion partners being a B or a T lymphocyte
    • 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
    • C07K2317/50—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
    • C07K2317/70—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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the invention relates to the field of antibody engineering, in particular to an anti-human IL-36R monoclonal antibody and application thereof, in particular to a mouse-derived monoclonal antibody with high affinity for human IL-36R and cross-reaction with monkey IL-36R Antibodies, chimeric antibodies, humanized antibodies and their applications.
  • Interleukin 36 or IL-36, including IL-36 ⁇ , IL-36 ⁇ , and IL-36 ⁇ (also called IL-1F6, IL-1F8, and IL-1F9), is a member of the interleukin 1 (IL-1) family, which is affected by The antibody is IL-36R (also known as IL-1RL2 or IL-1Rrp2).
  • IL-36R also known as IL-1RL2 or IL-1Rrp2
  • the interleukin-1 (IL-1) family is composed of 11 different ligands, namely IL-1 ⁇ (also called IL-1F11), IL-1 ⁇ (IL-1F2), IL-1 receptor antagonists (IL-1F3 or IL-1Ra), IL-18 (IL-1F4), IL-1F5 to IL-1F10 and IL-1F11 (ie IL-33) IL-36Ra (also known as IL-1F5) are natural antagonists of IL-36R.
  • IL-36 plays an important role in various inflammatory diseases such as psoriasis, inflammatory bowel disease, arthritis, systemic lupus erythematosus, and lung disease.
  • IL36 receptor antagonist (IL36 receptor antagonist, IL36Ra) is its natural antagonist. After extracellular IL-36 binds to the transmembrane protein IL-36R, it recruits IL-1 receptor accessory protein (IL-1 receptor accessory protein, IL-1RAcP) to form a heterotrimeric complex, which passes through the intracellular domain. The functional domain binds to the adaptor protein myeloid differentiation protein 88 (MyD88), activates the MAPKs/JNK/ERK signaling cascade, and initiates the expression of target genes through the nuclear transcription factor NFKB, thereby exerting a pro-inflammatory effect. In contrast, IL-36Ra competes with IL-36a, IL-36b and IL-36y for binding to IL-36R. The complex of IL-36Ra combined with IL-36R cannot recruit IL-1RAcP to bind, so it cannot activate the signal transduction pathway and play an anti-inflammatory effect.
  • MyD88 adaptor protein myeloid differentiation protein 88
  • the binding of anti-human IL-36R to IL-36R on the cell membrane easily stimulates effector activity to aggravate local inflammation and damage, and to a certain extent affects or even offsets the therapeutic effect of anti-human IL-36R antibody on inflammation.
  • mouse IL-36 does not interact with human IL-36 receptors, making animal modeling difficult and animal experiments difficult to effectively simulate the treatment of human patients. Therefore, the anti-IL-36R antibody needs to be further studied.
  • the present invention aims to solve at least one of the technical problems existing in the prior art.
  • the present disclosure provides an anti-human IL-36R monoclonal antibody and applications thereof.
  • the mouse-derived anti-human IL-36R monoclonal antibody was prepared by hybridoma technology, and the mouse-derived anti-human IL-36R antibody was screened to obtain an antibody that can specifically bind to human IL-36R and cross-react with cynomolgus monkey IL-36R.
  • a human-mouse chimeric anti-human IL-36R monoclonal antibody was constructed, and its binding to cell surface IL-36R was detected.
  • the humanized anti-human IL-36R monoclonal antibody was constructed by CDRs transplantation technology and CDR region mutation design, and the binding activity, affinity, and cross-reactivity of IL-36R between species were analyzed.
  • the humanized anti-human IL-36R monoclonal antibody can inhibit the secretion of inflammatory cytokines induced by IL36.
  • the present invention provides an anti-human IL-36R antibody or fragment thereof.
  • the human IL-36R antibody or its fragment can specifically bind to human IL-36R and monkey IL-36R, and the binding ability to human IL-36R is higher than the binding ability to monkey IL-36R.
  • candidate antibodies with specific binding ability to both human IL-36R and monkey IL-36R were selected during the hybridoma screening process.
  • the specificity of monkey IL-36R is also taken into account, and the humanized anti-human IL-36R antibody can be directly applied to the animal model of monkey skin inflammation for clinical evaluation of the therapeutic effect.
  • the anti-human IL-36R antibody or its fragment has no specific binding to murine IL-36R, and no specific binding to cell receptors of other members of the IL-1 family.
  • the KD value of the anti-human IL-36R antibody or its fragment binding to human IL-36R is ⁇ 5 ⁇ 10 9 .
  • the antibody or fragment thereof has a strong affinity for human IL-36R.
  • the anti-human IL-36R antibody comprises: a heavy chain variable region, the heavy chain variable region (VH) comprises antigenic determining regions (CDRs) 1, 2 and 3, wherein the VH CDR1 comprises Amino acid sequence at least 75% identical to the selected VH CDR1 amino acid sequence; VH CDR2 comprising an amino acid sequence at least 75% identical to the selected VH CDR2 amino acid sequence; VH CDR3 at least 75% identical to the selected VH CDR3 amino acid sequence
  • the amino acid sequence of identity specifically, can be at least 80, 83, 85, 88, 90, 93, 95, 98 and 99% identical; light chain variable region, the light chain variable region (VL) Including CDRs 1, 2 and 3, wherein, the VL CDR1 comprises an amino acid sequence with at least 75% identity to the selected VL CDR1 amino acid sequence, specifically, can be at least 80, 83, 85, 88, 90, 93, 95, 98 and more than 99% identity
  • amino acid sequences of the selected VH CDRs 1, 2 and 3 and the amino acid sequences of the selected VL CDRs 1, 2 and 3 are selected from one of the following:
  • the amino acid sequences of the selected VH CDRs 1 and 3 are shown in SEQ ID NOs: 27 and 30, respectively, the VH CDR2 has the amino acid sequence shown in GEIFPNX 1 GR, and the amino acids of the selected VL CDRs 1, 2 and 3 The sequences are shown in SEQ ID NOs: 31, 32 and 33;
  • VH CDRs 1, 2 and 3 are shown in SEQ ID NOs: 34, 35 and 36, respectively, and the amino acid sequences of the selected VL CDRs 1 and 2 are shown in SEQ ID NOs: 37 and 38. shown, VL CDR3 has the amino acid sequence shown in QQYX 2 X 3 YPLT,
  • X 1 is S or T
  • X 2 is N or T
  • X3 is S or T.
  • the anti-human IL-36R antibody or its fragment according to the embodiment of the present invention can bind to IL-36R on the surface of target cell membrane to stimulate effector activity, kill target cells through ADCC or CDC effect, thereby releasing inflammatory cytokines, aggravating local inflammation and injury.
  • the anti-human IL-36R antibody of the embodiment of the present invention adopts the heavy chain constant region of the IgG1 subtype and performs mutation transformation on it, namely Maintaining the stability of the IgG1 subtype antibody eliminates or reduces effector activity.
  • the VH comprises an amino acid sequence with at least 75% identity to SEQ ID NO: 5, specifically, may be at least 80, 83, 85, 88, 90, 93, 95, 98 and 99% identical
  • the VL includes an amino acid sequence that is at least 75% identical to SEQ ID NO: 11, specifically, can be at least 80, 83, 85, 88, 90, 93, 95, 98 and 99% identical. sex.
  • the VH comprises an amino acid sequence with at least 75% identity to SEQ ID NO: 15, specifically, may be at least 80, 83, 85, 88, 90, 93, 95, 98 and 99% identical
  • the above identity and the VL includes an amino acid sequence with at least 75% identity to SEQ ID NO: 18, in particular, may be at least 80, 83, 85, 88, 90, 93, 95, 98 and 99% more identical .
  • the VH comprises the amino acid sequence shown in SEQ ID NO: 1, 5, 6, 7, 8, 9 or 10
  • the VL comprises SEQ ID NO: 2, 11, 12, 13 or The amino acid sequence shown in 14.
  • the VH includes the amino acid sequence shown in SEQ ID NO: 3, 15, 16 or 17, and the VL includes SEQ ID NO: 4, 18, 19, 20, 21, 22, 23, The amino acid sequence shown in 24, 25 or 26.
  • amino acid sequence with at least 75% identity can be an amino acid sequence with 0, 1, 2 or more amino acid insertions, deletions or substitutions with the original sequence.
  • the VH includes the amino acid sequence shown in SEQ ID NO: 1, 5, 6, 7, 8, 9 or 10
  • the VL includes SEQ ID NO: 2, 11, 12, The amino acid sequence shown in 13 or 14.
  • the VH includes the amino acid sequence shown in SEQ ID NO: 3, 15, 16 or 17, and the VL includes SEQ ID NO: 4, 18, 19, 20, 21, 22, 23, The amino acid sequence shown in 24, 25 or 26.
  • HCDR1 with the amino acid sequence shown in SEQ ID NO: 27, HCDR2 with the amino acid sequence shown in any of SEQ ID NO: 28-29, and HCDR3 with the amino acid sequence shown in SEQ ID NO: 30 and has the LCDR1 of the amino acid sequence shown in SEQ ID NO:31, the LCDR2 of the amino acid sequence shown in SEQ ID NO:32, and the LCDR3 of the amino acid sequence shown in SEQ ID NO:33; or
  • HCDR1 with amino acid sequence shown in SEQ ID NO:34
  • HCDR2 with amino acid sequence shown in SEQ ID NO:35
  • HCDR3 with amino acid sequence shown in SEQ ID NO:36
  • LCDR1 with amino acid sequence shown in SEQ ID NO:37
  • LCDR2 of the amino acid sequence shown in SEQ ID NO: 38
  • LCDR3 of the amino acid sequence shown in any of SEQ ID NO: 39-40.
  • the present invention has a heavy chain variable region selected from any amino acid sequence in SEQ ID NO: 1, 5-10, and is selected from any amino acid sequence in SEQ ID NO: 2, 11-14 the indicated light chain variable region; or
  • It has a heavy chain variable region selected from the amino acid sequence shown in any of SEQ ID NO: 3 and 15-17, and is selected from the light chain variable region shown in any amino acid sequence of SEQ ID NO: 4 and 18-26.
  • the anti-human IL-36R antibody or fragment thereof can block the release of IL36R+ cells inflammatory cytokines induced by IL36 ⁇ , IL36 ⁇ , and/or IL36 ⁇ , and has no effector activity or significant effector activity decrease, wherein the inflammatory cytokine includes IL-8 and the effector activity includes ADCC and CDC.
  • the antibodies include mouse-derived antibodies, rabbit-derived antibodies, human-derived antibodies, chimeric antibodies, etc.; the antibody parts include Fab, Fab', F(ab')2, Fv, scFv, dAb, etc.
  • the present invention provides a humanized anti-human IL-36R antibody or a fragment thereof.
  • the humanized anti-human IL-36R antibody or its fragment is obtained by grafting CDRs on the basis of the aforementioned anti-human IL-36R antibody or its fragment of the present invention.
  • the humanized anti-human IL-36R antibody or a fragment thereof has a heavy chain constant region of IgG1 subclass, and the light at positions 234 and 235 of the heavy chain constant region of said IgG1 subclass Amino acids (L) were mutated to alanine (A).
  • the present invention provides a polynucleotide.
  • the polynucleotide encodes an anti-human IL-36R antibody or a fragment thereof according to any one of the foregoing, or a humanized anti-human IL-36R antibody or a humanized anti-human IL-36R antibody according to any one of the foregoing of the present invention. Fragment.
  • the present invention provides a construct, according to an embodiment of the present invention, the construct comprising the aforementioned polynucleotide.
  • the present invention provides a host cell, according to an embodiment of the present invention, the host cell comprises the aforementioned polynucleotide or the aforementioned nucleic acid construct.
  • the present invention provides a composition, according to an embodiment of the present invention, the composition comprises the anti-human IL-36R antibody or fragment thereof according to any one of the foregoing, or the humanized antibody described in any of the foregoing of the present invention.
  • the present invention provides a composition.
  • the composition comprises two or more anti-human IL-36R monoclonal antibodies, the two or more anti-human IL-36R monoclonal antibodies are selected from any one of the aforementioned An anti-human IL-36R antibody or a fragment thereof, the humanized anti-human IL-36R antibody or a fragment thereof of any one of the foregoing; and the two or more anti-human IL-36R monoclonal antibodies are directed against each other Human IL-36R has no competitive binding relationship.
  • the present invention provides a method for preparing an anti-human IL-36R antibody or a fragment thereof. According to an embodiment of the present invention, the method includes:
  • the present invention provides any one of the aforementioned anti-human IL-36R antibodies or fragments thereof, any one of the aforementioned humanized anti-human IL-36R antibodies or fragments thereof, the aforementioned polynucleotides, and the aforementioned constructs , the aforementioned host cells and the application of any one of the aforementioned compositions in the preparation of a medicament for the treatment of inflammatory diseases.
  • the inflammatory disease is an IL36-related inflammatory disease
  • the drug plays a role in the treatment of inflammatory diseases by blocking the induction of IL36 ⁇ , IL36 ⁇ , and/or IL36 ⁇ from binding to the cellular receptor IL36R. effect.
  • the inflammatory diseases include dermatitis, psoriasis, inflammatory bowel disease, arthritis, systemic lupus erythematosus, inflammatory lung disease, and chronic kidney disease; wherein psoriasis includes pustular type Psoriasis, generalized psoriasis, and palmoplantar pustulosis.
  • the inflammatory disease has symptoms of skin damage, and the drug improves skin damage by blocking IL36 ⁇ , IL36 ⁇ , and/or IL36 ⁇ -induced binding to the cellular receptor IL36R.
  • the present invention provides a method of treating a subject suffering from cancer or an inflammatory disease.
  • the method comprises administering to the subject a therapeutically effective amount of a composition, which is the aforementioned composition.
  • the inflammatory diseases include dermatitis, psoriasis, inflammatory bowel disease, arthritis, systemic lupus erythematosus, inflammatory lung disease and chronic kidney disease.
  • the psoriasis includes pustular psoriasis, generalized psoriasis and palmoplantar pustulosis.
  • the cancer is breast cancer.
  • the present invention provides a method for treating or preventing an IL-36R mediated disease in an individual in need thereof.
  • the method comprises administering a therapeutically effective amount of the aforementioned composition, optionally in combination with another therapeutic agent.
  • IL-36R is a membrane protein that interacts with IL-36 on the cell surface and is capable of binding to IL-36 ⁇ , IL-36 ⁇ , IL-36 ⁇ , and IL-36Ra.
  • IL-36 ⁇ , IL-36 ⁇ and IL-36 ⁇ are IL-36R receptor agonists
  • IL-36Ra is IL-36R receptor antagonist.
  • inactivating mutations in the gene encoding IL-36Ra lead to dysregulation of IL-36R signaling, mainly in diffuse pustular psoriasis, suggesting an important role for IL-36 in skin inflammation in psoriasis.
  • Studies have shown that IL-36 also has an important role in inflammatory diseases of other organs.
  • the term "specific binding” refers to a non-random binding reaction between two molecules, such as between an antibody and the antigen to which it is directed.
  • immunological binding refers to the specific binding reaction that occurs between an antibody molecule and an antigen for which the antibody is specific.
  • KD equilibrium dissociation constant
  • the strength or affinity of an immune-binding interaction can be expressed as the equilibrium dissociation constant (KD) of the interaction, where a smaller KD value indicates a higher affinity.
  • KD equilibrium dissociation constant
  • the immunological binding properties between two molecules can be quantified using methods well known in the art. One method involves measuring the rate of antigen binding site/antigen complex formation and dissociation.
  • association rate constants Ka or Kon
  • dissociation rate constants Kd or Koff
  • association rate constants Ka or Kon
  • dissociation rate constants Kd or Koff
  • concentrations concentrations and the actual rates of association and dissociation, see Malmqvist M, 1993, Nature, 361:186-187.
  • the ratio of Kd/Ka is equal to the dissociation constant KD, see Davies DR et al., 1990, Annual Rev Biochem., 59:439-473.
  • KD, Ka and Kd values can be measured by any effective method.
  • the dissociation constant is measured using bioluminescence interferometry.
  • dissociation constants can be measured using surface plasmon resonance techniques (eg, Biacore) or KinExa.
  • antibody herein is intended to include full-length antibodies and any antigen-binding fragments (ie, antigen-binding portions) or single chains thereof.
  • Full-length antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains linked by disulfide bonds.
  • Each heavy chain consists of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region.
  • the heavy chain constant region consists of three domains, namely CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (abbreviated as VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • the VH and VL regions can also be divided into hypervariable regions called complementarity determining regions (CDRs) separated by more conserved framework region (FR) regions.
  • CDRs complementarity determining regions
  • FR conserved framework region
  • Each VH and VL consists of three CDRs and four FRs, which are arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the constant regions of antibodies can mediate the binding of immunoglobulins to host tissues or factors, including various immune system cells (eg, effector cells) and the first component (Clq) of the traditional complement system.
  • monoclonal antibody or “monoclonal antibody” or “monoclonal antibody composition” refers to a preparation of antibody molecules of single molecular composition. Monoclonal antibodies are composed to exhibit a single binding specificity and affinity for a particular epitope.
  • an "antigen-binding fragment” (or simply antibody fragment) of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. It has been demonstrated that the antigen-binding function of antibodies can be performed by fragments of full-length antibodies.
  • binding fragments included in the "antigen-binding portion" of an antibody include (i) Fab fragments, monovalent fragments composed of VL, VH, CL and CH1; (ii) F(ab')2 fragments, comprising hinge region II Bivalent fragment of two Fab fragments linked by a sulfur bridge; (iii) Fd fragment composed of VH and CH1; (iv) Fv fragment composed of antibody one-arm VL and VH; (v) dAb fragment composed of VH ( Ward et al., (1989) Nature 341:544-546); (vi) isolated complementarity determining regions (CDRs); and (vii) Nanobodies, a single variable domain and two constant domain Heavy chain variable region.
  • the two domains of the Fv fragment, VL and VH are encoded by different genes, they can be linked by recombinant methods via a synthetic linker that makes the two into a single protein chain in which the VL and VH domains pair to form a monovalent molecule (called Single chain Fc (scFv); see eg Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).
  • scFv Single chain Fc
  • These single chain antibodies are also intended to be included within the meaning of the term.
  • These antibody fragments can be obtained by common techniques known to those skilled in the art, and the fragments can be screened for function in the same manner as intact antibodies.
  • Antigen-binding fragments of the present invention include those capable of specifically binding antigenic molecules.
  • Examples of antibody binding fragments include, for example, but not limited to, Fab, Fab', F(ab') 2 , Fv fragments, single chain Fv (scFv) fragments, and single domain fragments.
  • Fab fragments contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region.
  • Fab' fragments are generated by cleavage of the disulfide bond at the hinge cysteine of the F(ab')2 pepsin digest. Additional chemical conjugation of antibody fragments is known to those of ordinary skill in the art.
  • Fab and F(ab')2 fragments lack the crystallizable (Fc) region of intact antibodies, are cleared more rapidly from the animal's circulation, and may have less nonspecific tissue binding than intact antibodies (see e.g., Wahl et al. Man, 1983, J. Nucl. Med. 24:316).
  • an "Fc” region is a fragment crystallizable constant region of an antibody that does not contain an antigen-specific binding region.
  • the Fc region consists of two identical protein fragments, derived from the second and third constant domains (CH2 and CH3 domains, respectively) of the two heavy chains of the antibody.
  • the IgM and IgE Fc regions contain three heavy chain constant domains (CH2, CH3 and CH4 domains) in each polypeptide chain.
  • humanized antibody mainly refers to the re-expressed antibody of mouse monoclonal antibody transformed by gene cloning and DNA recombination technology, most of its amino acid sequence is replaced by human source sequence, and basically retains the parental mouse monoclonal antibody Affinity and specificity, and reduce its heterogeneity.
  • chimeric antibody means that a portion of the heavy and/or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to or homologous to the corresponding sequence derived from another antibody class or subclass.
  • Corresponding sequences in antibodies of one species or belonging to another class or subclass of antibodies are identical or homologous, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (US Patent No. 4,816,567; Morrison SL et al., Proc. . Natl. Acad. Sci. USA, 81:6851-6855, 1984).
  • chimeric antibody can include antibodies (eg, human-mouse chimeric antibodies) in which the heavy and light chain variable regions of the antibody are derived from a primary antibody (eg, a murine antibody) and the heavy and The light chain constant region is derived from a second antibody (eg, a human antibody).
  • a primary antibody eg, a murine antibody
  • a second antibody eg, a human antibody
  • the heavy chain (heavy chain, H chain) is about twice the size of the light chain, contains 450-550 amino acid residues, and has a molecular weight of about 55 or 75 kD.
  • Each H chain contains a cyclic peptide composed of 4 to 5 intrachain disulfide bonds. Different H chains have different antigenicity due to the arrangement sequence of amino acid composition, the number and position of disulfide bonds, and the types and quantities contained.
  • H chain antigenicity According to the difference in H chain antigenicity, they can be divided into 5 categories: ⁇ chain , ⁇ chain, ⁇ chain, ⁇ chain and ⁇ chain, different H chain and L chain ( ⁇ or ⁇ chain) make up the complete immunoglobulin molecules are called IgM, IgG, IgA, IgD and IgE respectively.
  • the ⁇ , ⁇ and ⁇ chains contain 4 peptides, and the ⁇ and ⁇ chains contain 5 cyclic peptides.
  • light chain refers to a polypeptide chain that is smaller in molecular weight relative to the heavy chain in an immunoglobulin monomer molecule.
  • the variable region (VL) of the amino acid composition sequence in the region near the amino terminal (N-terminal) 1/2 of each light chain is the light chain variable region (VL), which is an integral part of the Ig molecule and the antigen binding site.
  • the amino acid composition and arrangement sequence in the remaining half of the region are relatively stable in the light chain constant region (CL). Due to some differences in the amino acid sequence within the constant region of the light chain, there are two types of light chains, ⁇ and ⁇ .
  • germline also known as the germline, is the antibody-forming cell that has the complete set of genes encoding Ig molecules (i.e. a limited number of C genes and an unknown number of V genes, which are formed through long-term evolution and are derived from parental cells through germ cells) Passed on to offspring, the heavy chain gene of human immunoglobulin is encoded by V-D-J-C gene fragment; the light chain gene is encoded by V-J-C gene fragment, and the number of gene fragments varies.
  • the human heavy chain gene is located on chromosome 14
  • the long arm spanning about 1,100kb, is composed of four gene fragments V, D, J and C, including about 95 Va gene fragments (divided into seven families of VH1 ⁇ VH7, of which 65 functional gene fragments and 27 D genes) fragment, 6 JH gene fragments and 9 CH gene fragments.
  • the Va gene fragment is located upstream
  • the D gene fragment is located between the VH and JH gene clusters
  • the JH gene is located downstream of DH, and is separated from the downstream C gene region by about 7Kb.
  • Cluster arrangement spanning about 200kb, P and S genes are located immediately downstream of the JH gene segment, and C8 downstream is followed by C ⁇ , C ⁇ and C ⁇ .
  • Human light chain genes are divided into ⁇ and x genes, which are located on chromosome 22, respectively. Arm and short arm of chromosome 2. There are about 40 functional VK gene fragments, 5 functional J and 1 C ⁇ after the Vc gene fragment; about 30 V ⁇ gene fragments, 4 J ⁇ gene fragments and 4 C ⁇ genes Fragment.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked.
  • plasmid refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector in which additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) can integrate into the genome of the host cell after introduction into the host cell, and thereby replicate together with the host genome.
  • vectors are capable of directing the expression of the genes to which they are operably linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply “expression vectors”).
  • expression vectors useful in recombinant DNA technology typically exist in the form of plasmids.
  • viral vectors eg, replication-defective retroviruses, adenoviruses and adeno-associated viruses
  • polynucleotide is intended to include DNA molecules and RNA molecules. Polynucleotides can be single-stranded or double-stranded, and can be cDNAs or fragments thereof.
  • polypeptide refers to a chain comprising at least two consecutively linked amino acid residues, with no upper limit on the length of the chain.
  • One or more amino acid residues in a protein may contain modifications such as, but not limited to, glycosylation, phosphorylation, or disulfide bonds.
  • a “protein” may comprise one or more polypeptides.
  • host cell is a cell in which a vector can be propagated and its DNA can be expressed, which cell can be a prokaryotic cell or a eukaryotic cell.
  • the term also includes any progeny of the subject host cell. It should be understood that not all progeny are identical to the parental cell, and such progeny are included due to the possibility of mutation during replication.
  • the anti-human IL-36R antibody or fragment thereof of the embodiment of the present invention has at least one of the following beneficial technical effects:
  • the anti-human IL-36R antibody of the embodiment of the present invention not only has a high affinity for human IL36R, but also can inhibit IL- 36-induced inflammatory cytokine secretion, so it is expected to have a better therapeutic effect in the treatment of inflammatory diseases, especially inflammatory-related skin diseases such as psoriasis.
  • the anti-human IL-36R antibody or its fragment of the embodiment of the present invention has a good therapeutic effect on cancer and inflammatory diseases, and the antibody or its fragment takes into account the adaptability of animal models and the effectiveness of animal tests, and is suitable for hybridization.
  • candidate antibodies with specific binding ability to both human IL-36R and monkey IL-36R were selected, and the specificity to monkey IL-36R was also taken into account in the process of humanization transformation.
  • the humanized anti-human IL-36R antibody can be directly administered to the monkey skin inflammation animal model for clinical evaluation of the therapeutic effect.
  • anti-human IL-36R antibody can bind to IL-36R on the surface of target cell membrane to stimulate effector activity, kill target cells through ADCC or CDC effect, thereby releasing inflammatory cytokines, aggravating local inflammation and injury.
  • the IgG4 subtype antibody has low effector activity but poor stability, and the anti-human IL-36R antibody of the present invention adopts the heavy chain constant region of the IgG1 subtype and mutates it, which maintains the stability of the IgG1 subtype antibody. Effector activity is eliminated or reduced.
  • Figure 1 Schematic diagram of the results of the binding activity of the murine monoclonal antibody of the embodiment of the present invention to the hu-IL36R protein;
  • Figure 2 Schematic diagram of the results of the binding activity of the human-mouse chimeric antibody of the embodiment of the present invention to hu-IL36R and cyno-IL36R on the surface of recombinant HEK293 cells, wherein A is the human-mouse chimeric antibody and hu-IL36R on the surface of recombinant HEK293 cells
  • the binding curve of , B is the binding curve of human-mouse chimeric antibody and recombinant HEK293 cell surface cyno-IL36R;
  • Figure 3 Schematic diagram of the results of the inhibitory activity of the human-mouse chimeric antibody on IL-36-induced inflammatory cytokine secretion in the Jurkat-hIL36R-IL1Racp-NF- ⁇ B cell model according to the embodiment of the present invention
  • Figure 4 Schematic diagram of the results of the inhibitory activity of human-mouse chimeric antibodies on IL-36-induced inflammatory cytokine secretion in epidermal squamous cell carcinoma A431 cells according to the embodiment of the present invention, wherein A is the chimeric antibody pair in A431 cells Schematic diagram of the results of the inhibitory activity of IL-36 ⁇ -induced IL-8 secretion, B is the result of the inhibitory activity of the chimeric antibody on IL-36 ⁇ -induced IL-8 secretion in A431 cells, and C is the result of the chimeric antibody in A431 cells Schematic diagram of the results of the inhibitory activity of the antibody on IL-36 ⁇ -induced IL-8 secretion,
  • Figure 5 Schematic diagram of the results of the binding of humanized antibodies to human IL36R and cynomolgus monkey IL36R according to the embodiment of the present invention, wherein, A is a schematic diagram of the results of binding of humanized antibodies to human IL36R, and B is a schematic diagram of the results of binding of humanized antibodies to cynomolgus monkeys Schematic diagram of the results of binding of IL36R;
  • Figure 6 Schematic diagram of the results of the inhibitory activity of humanized antibodies on IL-36-induced inflammatory cytokine secretion in epidermal squamous cell carcinoma A431 cells according to the embodiment of the present invention, wherein A is the effect of humanized antibodies on IL-36 in A431 cells Schematic diagram of the results of the inhibitory activity of -36 ⁇ -induced IL-8 secretion, B is the schematic diagram of the results of the inhibitory activity of the humanized antibody on IL-36 ⁇ -induced IL-8 secretion in A431 cells, and C is the result of human-derived antibodies in A431 cells Schematic representation of the results of the inhibitory activity of IL-36 ⁇ -induced IL-8 secretion by IL-8 antibodies.
  • first and second are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implying the number of indicated technical features. Thus, a feature defined as “first” or “second” may expressly or implicitly include one or more of that feature. Further, in the description of the present invention, unless otherwise specified, "plurality" means two or more.
  • Example 1 Preparation and screening of hybridoma cells with anti-human IL-36R monoclonal antibody
  • the obtained human interleukin 36 receptor protein (with His tag, article number CI17, Nearshore Protein Technology Co., Ltd.) was subjected to conventional methods (Lonberg, N., et al., nature 368 (1994) 856-859; Fishwild, D.M., et al., Nat. Biotechnol. 14 (1996) 845-851 and WO 98/24884) immunized 10 Balb/c mice.
  • the protein dose for the initial immunization of each mouse was 50ug, and then three booster immunizations were performed, and the protein dose was 25ug/mouse.
  • IL-36R Interleukin 36 receptor protein
  • Fc tag Fc tag
  • product number CJ62 near the concentration of 1ug/ml
  • Anshore Protein Technology Co., Ltd. was coated in a 96-well plate, 100 ⁇ l per well, incubated overnight at 4°C, then washed off the coating solution, added 200ul of protein-free blocking buffer to each well, and allowed to stand at room temperature for 2 hours.
  • Mouse serum was pre-diluted 200-fold in PBSA (1% BSA in PBS) and serially diluted 1:2 in 12 gradients to 409,600-fold.
  • mice serum After washing off the blocking solution, add the diluted mouse serum to a 96-well plate, incubate at room temperature for 1 hour, wash the 96-well with PBST, and then add 100ul of secondary antibody (HRP-labeled goat anti-mouse IgG) diluted 1:3000 to each well. , stand at room temperature for 45 minutes, wash 5 times with PBST, add 50ul of TMB equilibrated at room temperature to each well, incubate at room temperature for 10 minutes, and measure the absorbance at 450 nm.
  • secondary antibody HRP-labeled goat anti-mouse IgG
  • mice with higher serum titers were taken, the spleen was taken, the mouse B cells were isolated, mixed with pre-prepared mouse myeloma cells P3X63Ag8.653 in a ratio of 1:2, and electroshock fusion was performed; Hybridoma cells were inoculated into 384-well plates and cultured by adding appropriate medium. After 7-10 days, the culture supernatant of hybridoma cells was taken, and positive clones were screened by the above enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • RNAfast200 kit (Shanghai Feijie Biotechnology Co., Ltd.); Total RNA of tumor cells was reverse transcribed into cDNA; degenerate primers (Anke Krebber.
  • the heavy chain variable region of the above-mentioned murine anti-human IL-36R monoclonal antibody and the heavy chain constant region of the published human monoclonal antibody IgG1 subclass (with two substitution mutations Leu234Ala and Leu235Ala, thereby reducing effector function)
  • spliced together to form the heavy chain of chimeric antibody (hIgG1-LALA)
  • the heavy chain was constructed into mammalian cell expression vector
  • murine anti-human IL-36R The variable region of the light chain of the monoclonal antibody and the constant region of the light chain of the published human monoclonal antibody Kappa subclass are spliced together to form the light chain of the chimeric antibody, and the light chain is constructed into a mammalian cell expression vector.
  • the heavy chain plasmid and light chain plasmid of the constructed anti-human IL-36R chimeric antibody were paired and mixed, and HEK293 cells were transfected with polyethyleneimine (PEI).
  • PEI polyethyleneimine
  • concentration, volume and total protein amount of chimeric antibody protein were detected. The results are shown in Table 2.
  • Example 3 Activity and physicochemical properties of anti-human IL-36R chimeric antibodies
  • IL-36R Encoding human IL-36R (huIL-36R, NCBI accession: NM_001351446), mouse IL-36R (muIL-36R, NCBI accession: NM_001356478) and cynomolgus monkey IL-36R (cyno-IL36R, NCBI accession: XP_014968513), respectively
  • the plasmid of the long gene was transfected into HEK293 cells with polyethyleneimine (PEI), and single cells with stable and high expression were selected for future use.
  • PEI polyethyleneimine
  • the HEK293 cells expressing human IL-36R obtained above were plated in a 96-well plate at 30,000/well, and then 50ul of the chimeric antibody obtained above was added to each well, starting from 100 nM, and adding 1 :3 serial dilutions of 8 gradients followed by standing at room temperature for 1 hour. Wash with PBSA (PBS solution containing 1% BSA), add 50ul 1:400 diluted secondary antibody (FITC-goat anti-human IgG), let stand at 4°C for 30 minutes, then wash with PBSA, and finally resuspend cells with 50ul PBSA, The mean fluorescence intensity of the target cell population was analyzed by flow cytometry.
  • PBSA PBS solution containing 1% BSA
  • FITC-goat anti-human IgG FITC-goat anti-human IgG
  • HEK293 cells expressing mouse IL-36R and cynomolgus monkey IL-36R were also plated in a 96-well plate, and then the binding of the chimeric antibody to these two cells was detected according to the above procedure.
  • Table 3a EC50 values of chimeric antibodies that bind to human IL-36R on the surface of HEK293 cells
  • Table 3b EC50 values of chimeric antibodies that bind to monkey IL-36R on the surface of HEK293 cells).
  • the binding kinetic parameters of the chimeric antibody to the antigen huIL-36R were analyzed by Fortebio (BLITZ pro1.1.0.28) instrument. Before the measurement, soak the AHC biological probe in PBS for 10 minutes; then place the probe in PBS containing 100nM antibody for 300 seconds to capture the antibody with hIgG1-Fc tag; further bind the probe to 100nM antigen The reaction time was 400 seconds for binding; then the probe was transferred to PBS, and the dissociation reaction was performed for 600 seconds. After the experiment was completed, the response value of the blank parent was deducted, and the 1:1 Langmuir binding mode was fitted with the software to calculate the kinetic constant of antigen-antibody binding. The results are shown in Table 4.
  • the parameters of the binding kinetics of the chimeric antibody to human IL-1R1 were also analyzed by Fortebio (BLITZ pro1.1.0.28) instrument, and the binding signal was shown as 0 (results not shown). This indicates that all chimeric antibodies specifically bind human IL-36R. Therefore, the results in Table 4 indicate that the purified chimeric antibody can specifically bind to human IL-36R with high affinity in vitro.
  • Cross-competitive binding of chimeric antibodies to human IL-36R was determined using a Fortebio (BLITZ pro1.1.0.28) instrument.
  • human IL-36R His-tagged
  • PBSA 1% BSA in PBS
  • chimeric antibody 2 was added to determine whether chimeric antibody 2 competitively binds to human IL-36R.
  • the chimeric antibodies obtained above were tested for physical and chemical properties, including monomer rate (SEC), hydrophobicity (HIC), thermal stability (TM) and molecular weight determination (MALS).
  • SEC monomer rate
  • HIC hydrophobicity
  • TM thermal stability
  • MALS molecular weight determination
  • Antibody monomer rate analysis inject 50ul of antibody with a concentration of 1mg/ml into a pre-equilibrated chromatographic column (TSKgel G3000SWXL 7.8*300 (TOSHI), flow at room temperature for 35min at a flow rate of 0.7ml/min, and detect at the same time
  • TSKgel G3000SWXL 7.8*300 TOSHI
  • the absorption value of the machine WATERS UPLC CLASS ACQUITYH
  • A280 is used to determine the monomer content and proportion of the antibody according to the peak time and peak volume.
  • Antibody hydrophobic property analysis The hydrophobic property of the antibody was analyzed using a WATERS ARC instrument and a hydrophobic chromatography column (TSKgel Butyl-NPR (4.6mmX3.5cm, Cat No 14947) instructions for use.
  • TM Thermal stability of the antibody was analyzed using Bio-rad's real-time PCR instrument and ProteoSTAT Thermal shift assay kit (Cat No. ENZ-51027-K400).
  • IL-36R Three plasmids encoding human IL-36R (huIL-36R, NCBI accession: NM_001351446), encoding human IL-1RAcP and encoding Luc2P-NFkB gene were stably transfected into Jurkat cells in turn (Shanghai Cell Bank, Cat.No.TCHU123) , select single cells with stable and high expression for use, and establish Jurkat-hIL36R-IL1Racp-NF- ⁇ B reporter gene cell line.
  • the chimeric antibodies obtained above were formulated into three concentrations of 167nM, 10nM and 0.7nM, respectively, and mixed with IL-36R ligands with approximate EC50 concentrations (the three ligands of IL36 ⁇ , IL36 ⁇ and IL36 ⁇ had been previously determined to stimulate reporters.
  • the EC50 concentration of the fluorescent signal produced by the gene cells) was added to a white bottom transparent 96-well plate according to 50ul/well.
  • the reporter gene cells obtained above were centrifuged to change the medium, and then added to the above-mentioned white bottom transparent 96-well plate at 1 ⁇ 10 5 cells/well.
  • Anti-human IL-36R chimeric antibody inhibits IL-36-induced IL-8 secretion in epidermal squamous cell carcinoma A431 cells
  • A431 cells produced the cytokine IL-8 when stimulated by IL36R ligands (IL36 ⁇ , IL36 ⁇ and IL36 ⁇ ), while anti-IL-36R antibodies inhibited the production of IL-8 by A431 cells.
  • the chimeric antibody obtained above was diluted 3-fold in 10 gradients starting from 30ug/ml, and mixed with IL-36R ligands with an approximate EC50 concentration (the three ligands IL36 ⁇ , IL36 ⁇ and IL36 ⁇ were previously determined. The EC50 concentration of each body); the mixed solution was added to a white bottom transparent 96-well plate according to 50ul/well.
  • the A431 cells were centrifuged to change the medium, and then added to the above-mentioned white bottom permeable 96-well plate at 1x10 5 cells/well, cultured for 48 hours, and then using R&D Systems DUO-SET ELISA kit, according to the standard protocol provided by the manufacturer, by The levels of IL-8 in the cell supernatants were assessed by ELISA.
  • Example 4 Design and preparation of anti-human IL-36R humanized antibody
  • mouse anti-human IL-36R antibody obtained previously, that is, to change as many mouse-derived sequences as possible to human-derived sequences in the mouse antibody.
  • binding specificity, kinetic parameters, physicochemical properties, stability and other characteristics of murine antibodies should be preserved.
  • human framework sequences for mouse leader sequences were selected based on framework homology, CDR structure, conserved canonical residues, conserved interface packaging residues, and other parameters.
  • the formed humanized antibody including binding specificity, binding kinetic parameters, physicochemical properties, etc., selects the humanized antibody that shows better or equal binding compared with the parental murine antibody for subsequent further verification.
  • murine antibodies A10-6B12 and A19-35D19 were selected for humanization (cloned into human IgG1-LALA/ ⁇ backbone, see Table 7 for variable region sequences).
  • the heavy chain variable region of the above-mentioned humanized anti-human IL-36R monoclonal antibody and the heavy chain constant region of the published human monoclonal antibody IgG1 subclass are spliced together to form the heavy chain (hIgG1-LALA) of a humanized antibody, and the heavy chain is constructed into a mammalian cell expression vector;
  • the heavy chain variable region of the anti-IL-36R monoclonal antibody and the heavy chain constant region 1 (hIgG1-VH1) of the published human monoclonal antibody IgG1 subclass are spliced together to form the heavy chain Fd ( VH-CH1), the heavy chain Fd was also constructed into a mammalian cell expression vector.
  • the light chain variable region of the humanized anti-human IL-36R monoclonal antibody and the light chain constant region of the published human monoclonal antibody Kappa subclass are spliced together to form the light chain of the humanized antibody. Constructed into mammalian cell expression vectors.
  • the constructed anti-human IL-36R chimeric antibody heavy chain plasmid (including heavy chain Fd plasmid) and light chain plasmid were paired and mixed, and HEK293 cells were transfected with polyethyleneimine (PEI), and the cell supernatant was collected after about 7 days , using Protein A to purify to obtain the anti-human IL-36R chimeric antibody protein (the humanized antibody is of hIgG1-LALA subtype, and the Fab form of the partially humanized antibody hIgG1-LALA subtype is also obtained, see Table 8).
  • Example 5 Activity and function of anti-human IL-36R humanized antibodies
  • the binding kinetic parameters of the humanized antibody to the antigen huIL-36R were analyzed by Fortebio (BLITZ pro1.1.0.28) instrument. Before the measurement, soak the AHC biological probe in PBS for 10 minutes; then place the probe in PBS containing 100nM antibody for 300 seconds to capture the antibody with hIgG1-Fc tag; further bind the probe to 100nM antigen The reaction time was 400 seconds for binding; then the probe was transferred to PBS, and the dissociation reaction was performed for 600 seconds. After the experiment was completed, the response value of the blank parent was deducted, and the 1:1 Langmuir binding mode was fitted with the software to calculate the kinetic constant of antigen-antibody binding.
  • the binding activity of the antibody (Fabric format) to human IL-36R was also determined using the Fortebio (BLITZ pro1.1.0.28) instrument.
  • human IL-36R His-tagged
  • PBSA 1% BSA in PBS
  • the probe was further mixed with 100 nM
  • the antibody Fab underwent a binding reaction for 400 seconds; then the probe was transferred to PBS for a dissociation reaction for 600 seconds.
  • the response value of the blank parent was deducted, and the 1:1 Langmuir binding mode was fitted with the software to calculate the kinetic constant of antigen-antibody binding.
  • Table 9 shows the affinity detection results of the above-mentioned humanized antibodies and Fab antibodies.
  • binding kinetic parameters of the chimeric antibody to human IL-1R1 were also analyzed by Fortebio (BLITZ pro1.1.0.28) instrument, and the binding signal was shown as 0 (result not shown), which indicated that all humanized antibodies specifically bound Human IL-36R.
  • the HEK293 cells expressing human IL-36R obtained above were plated in a 96-well plate at 30,000/well, and then 50ul of the above-obtained humanized antibody was added to each well, starting from 100 nM, and Eight gradients were serially diluted 1:3 and then allowed to stand at room temperature for 1 hour. Wash with PBSA (PBS solution containing 1% BSA), add 50ul 1:400 diluted secondary antibody (FITC-goat anti-human IgG), let stand at 4°C for 30 minutes, then wash with PBSA, and finally resuspend cells with 50ul PBSA, The mean fluorescence intensity of the target cell population was analyzed by flow cytometry.
  • the HEK293 cells expressing mouse IL-36R and cynomolgus monkey IL-36R were also plated in 96-well plates, and the binding of the humanized antibody to these two cells was detected according to the above procedure.
  • Anti-human IL-36R humanized antibody inhibits IL-36-induced IL-8 secretion in A431 cells
  • A431 cells produced the inflammatory cytokine IL-8 when stimulated by IL36R ligands (IL36 ⁇ , IL36 ⁇ and IL36 ⁇ ), while IL-36R antibody inhibited the production of IL-8 by A431 cells.
  • the above-obtained humanized antibody was diluted 3-fold in 10 gradients starting from 30ug/ml, and mixed with IL-36R ligand of approximate EC50 concentration (the three IL36 ⁇ , IL36 ⁇ and IL36 ⁇ had been previously determined in advance. The respective EC50 concentrations of the ligands); the mixed solution was added to a white bottom transparent 96-well plate according to 50ul/well.
  • the A431 cells were centrifuged to change the medium, and then added to the above-mentioned white bottom permeable 96-well plate at 1x10 5 cells/well, cultured for 48 hours, and then using R&D Systems DUO-SET ELISA kit, according to the standard protocol provided by the manufacturer, by The levels of IL-8 in the cell supernatants were assessed by ELISA.
  • the results demonstrate that the humanized antibody can efficiently inhibit the secretion of the inflammatory cytokine IL-8 from A431 cells stimulated by IL-36R ligands (IL36 ⁇ , IL36 ⁇ and IL36 ⁇ ) in a dose-dependent manner.
  • the human IL-36R antibody of the embodiment of the present invention has a high affinity for human IL36R, and can inhibit the secretion of inflammatory cytokines induced by IL-36, and the antibody has a high affinity for human and monkey IL-36R. Has specific binding ability.
  • the human IL-36R antibody of the embodiment of the present invention adopts the heavy chain constant region of the IgG1 subtype and carries out mutational transformation, which not only maintains the stability of the IgG1 subtype antibody but also eliminates or reduces the effector activity, avoiding the need for anti-inflammatory drugs.
  • Human IL-36R antibody binds to the surface of target cell membrane IL-36R stimulates effector activity, releases inflammatory cytokines, aggravates local inflammation and damage, and makes the anti-inflammatory and anti-tumor effects of the human IL-36R antibody of the embodiment of the present invention better and safe Sex is higher.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Immunology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Cell Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Urology & Nephrology (AREA)
  • Dermatology (AREA)
  • Rheumatology (AREA)
  • Epidemiology (AREA)
  • Pain & Pain Management (AREA)
  • Pulmonology (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Hematology (AREA)
  • Developmental Biology & Embryology (AREA)

Abstract

提供了抗人IL-36R单克隆抗体及其应用。通过杂交瘤技术制备鼠源抗人IL-36R单克隆抗体，对鼠源抗人IL-36R抗体进行筛选获得能特异性结合人IL-36R并与食蟹猴IL-36R具有交叉反应的抗体，构建人-鼠嵌合抗人IL-36R单克隆抗体，检测其与细胞表面IL-36R结合实验。在鼠源抗体、嵌合抗体性能分析的基础上，采用CDRs移植技术、CDR区突变设计构建人源化抗人IL-36R单克隆抗体，分析结合活性、亲和力、种间IL-36R交叉反应，所述人源化抗人IL-36R单克隆抗体能抑制IL36诱导的炎性细胞因子分泌。

Description

抗人IL-36R抗体及其应用
优先权信息
本申请请求2021年2月8日向中国国家知识产权局提交的、专利申请号为202110183575.1的专利申请的优先权和权益，并且通过参照将其全文并入此处。
技术领域
本发明涉及抗体工程领域，具体涉及一种抗人IL-36R单克隆抗体及其应用，特别是涉及一种针对人IL-36R具有高亲和力、与猴IL-36R具有交叉反应的鼠源单克隆抗体、嵌合抗体、人源化抗体及其应用。
背景技术
白介素36，即IL-36，包括IL-36α，IL-36β和IL-36γ(也叫IL-1F6,IL-1F8和IL-1F9)，是白介素1(IL-1)的家族成员，其受体是IL-36R(也叫做IL-1RL2或IL-1Rrp2)。白介素1(IL-1)家族是由11个不同配体组成，即IL-1α(也叫做IL-1F11)、IL-1β(IL-1F2)、IL-1受体拮抗剂(IL-1F3或IL-1Ra)、IL-18(IL-1F4)、IL-1F5至IL-1F10和IL-1F11(即IL-33)IL-36Ra(也叫做IL-1F5)是IL-36R天然的拮抗剂。IL-36作为一种新发现的促炎因子,在银屑病、炎性肠病、关节炎、系统性红斑狼疮、肺部疾病等多种炎性疾病中发挥着重要作用。
IL36受体拮抗剂(IL36 receptor antagonist,IL36Ra)是其天然拮抗剂。细胞外的IL-36与跨膜蛋白IL-36R结合后，招募IL-1受体辅助蛋白(IL-1 receptor accessory protein,IL-1RAcP)，形成异源三聚体复合物，通过胞内区功能域与接头蛋白髓样分化蛋白88(MyD88)结合，活化MAPKs/JNK/ERK信号级联途径，通过核转录因子NFKB启动靶基因的表达，从而发挥促炎症作用。而IL-36Ra与IL-36a、IL-36b和IL-36y竞争性结合IL-36R，。IL-36Ra与IL-36R结合后的复合物不能招募IL-1RAcP结合，从而不能激活信号转导通路而起到抗炎症作用。
随着IL-36R对炎性疾病发生发展的作用机理日渐清晰，治疗性抗人IL-36R抗体吸引了越来越多的关注。我们经过研究发现现有技术存在以下不足：首先，抗人IL-36R抗体在抗炎、减少炎症细胞因子释放等方面的能力与其对人IL-36R的结合能力的相关性并不稳定，前期试验结果表明并非亲和力越高抗炎效果越好，抗人IL-36R抗体对炎症的治疗效果受到除亲和力以外其它因素的影响。其次，抗人IL-36R对细胞膜表面IL-36R的结合易激发效应子活性加重局部炎症和损伤，一定程度上影响甚至抵消了抗人IL-36R抗体对炎症的治疗效果。另外，小鼠IL-36不与人IL-36受体相互作用，动物造模困难、动物试验难以有效模拟对人类患者的治疗情况。由此，抗IL-36R抗体有待进一步研究。
发明内容
本发明旨在至少解决现有技术中存在的技术问题之一。本公开提供一种抗人IL-36R单克隆抗体及其应用。通过杂交瘤技术制备鼠源抗人IL-36R单克隆抗体，对鼠源抗人IL-36R抗体进行筛选获得能特异性结合人IL-36R并与食蟹猴IL-36R具有交叉反应的抗体，构建人-鼠嵌合抗人IL-36R单克隆抗体，检测其与细胞表面IL-36R结合实验。在鼠源抗体、嵌合抗体性能分析的基础上，采用CDRs移植技术、CDR区突变设计构建人源化抗人IL-36R单克隆抗体，分析结合活性、亲和力、种间IL-36R交叉反应，所述人源化抗人IL-36R单克隆抗体能抑制IL36诱导的炎性细胞因子分泌。
具体而言：
一方面，本发明提供一种抗人IL-36R抗体或其片段。根据本发明的实施例，该人IL-36R抗体或其片段能够特异性结合人IL-36R和猴IL-36R，并且对人IL-36R的结合能力高于对猴IL-36R的结合能力。
本发明实施例的抗人IL-36R抗体或其片段，在杂交瘤筛选过程中即选择了对人IL-36R、猴IL-36R均具有特异性结合能力的候选抗体，在人源化改造过程中也兼顾了对猴IL-36R的特异性，人源化抗人IL-36R抗体可以直接施用于猴皮肤炎症动物模型用于临床期评估治疗效果。
进一步，根据本发明的实施例，该抗人IL-36R抗体或其片段与鼠IL-36R无特异性结合，与IL-1家族其它成员的细胞受体无特异性结合。
进一步，该抗人IL-36R抗体或其片段与人IL-36R结合的KD值<5×10 9。由此，该抗体或其片段与人IL-36R的亲和力强。
通过分析抗体的氨基酸序列来鉴定抗体的CDR区的方法是公知的，并且CDR的许多定义是常用的。Kabat定义基于序列变异性，Chothia定义基于结构环区域的位置。除非在本公开内容中特别指出，否则在本公开内容中默认使用Kabat编号。
根据本发明的实施例，该抗人IL-36R抗体包括：重链可变区，所述重链可变区(VH)包括抗原决定区(CDRs)1、2和3，其中，VH CDR1包含与所选VH CDR1氨基酸序列具有至少75％同一性的氨基酸序列；VH CDR2包含与所选VH CDR2氨基酸序列具有至少75％同一性的氨基酸序列；VH CDR3与所选VH CDR3氨基酸序列具有至少75％同一性的氨基酸序列，具体地，可以是至少80、83、85、88、90、93、95、98和99％以上同一性；轻链可变区，所述轻链可变区(VL)包括CDRs 1、2和3，其中，VL CDR1包含与所选VL CDR1氨基酸序列具有至少75％同一性的氨基酸序列，具体地，可以是至少80、83、85、88、90、93、95、98和99％以上同一性；VL CDR2包含与所选VL CDR2氨基酸序列具有至少75％同一性的氨基酸序列，具体地，可以是至少80、83、85、88、90、93、95、98和99％以上同一性；VL CDR3与所选VL CDR3氨基酸序列具有至少75％同一性的氨基酸序列，具体地，可以是至少80、83、85、88、90、93、95、98和99％以上同一性。
根据本发明的一些实施例，所述所选VH CDRs 1、2和3的氨基酸序列以及所述所选VL CDRs 1、2和3的氨基酸序列选自下列之一：
所述所选VH CDRs 1和3的氨基酸序列分别如SEQ ID NO：27和30所示，VH CDR2具有GEIFPNX 1GR所示的氨基酸序列，且所述所选VL CDRs 1、2和3的氨基酸序列如SEQ ID NO：31、32和33所示；
所述所选VH CDRs 1、2和3的氨基酸序列分别如SEQ ID NO：34、35和36所示，且所述所选VL CDRs 1和2的氨基酸序列如SEQ ID NO：37和38所示，VL CDR3具有QQYX 2X 3YPLT所示的氨基酸序列，
其中，
X 1为S或T；
X 2为N或T；
X 3为S或T。
根据本发明实施例的抗人IL-36R抗体或其片段能结合到靶细胞膜表面IL-36R激发效应子活性，通过ADCC或CDC效应杀伤靶细胞，从而释放炎症细胞因子、加重局部炎症和损伤。根据本发明的实施例，由于IgG4亚型抗体效应子活性低但稳定性较差，本发明实施例的抗人IL-36R抗体采用IgG1亚型的重链恒定区并对其进行突变改造，即保持了IgG1亚型抗体的稳定性又消除或降低了效应子活性。
根据本发明的实施例，所述VH包括与SEQ ID NO：5具有至少75％同一性氨基酸序列，具体地，可以是至少80、83、85、88、90、93、95、98和99％以上同一性，且所述VL包括与SEQ ID NO：11具有至少75％同一性氨基酸序列，具体地，可以是至少80、83、85、88、90、93、95、98和99％以上同一性。
根据本发明的实施例，所述VH包括与SEQ ID NO：15具有至少75％同一性氨基酸序列，具体地，可以是至少80、83、85、88、90、93、95、98和99％以上同一性且所述VL包括与SEQ ID NO：18具有至少75％同一性氨基酸序列，具体地，可以是至少80、83、85、88、90、93、95、98和99％以上同一性。
根据本发明的实施例，所述VH包括SEQ ID NO:1、5、6、7、8、9或10所示的氨基酸序列，所述VL包括SEQ ID NO：2、11、12、13或14所示的氨基酸序列。
根据本发明的实施例，所述VH包括SEQ ID NO：3、15、16或17所示的氨基酸序列，所述VL包括SEQ ID NO：4、18、19、20、21、22、23、24、25或26所示的氨基酸序列。
其中，具有至少75％同一性氨基酸序列，可以是与原序列具有具有0、1、2或多个氨基酸插入、缺失或替换的氨基酸序列。
进一步，根据本发明的实施例，所述VH包括SEQ ID NO:1、5、6、7、8、9或10所示的氨基酸序列，所述VL包括SEQ ID NO：2、11、12、13或14所示的氨基酸序列。
根据本发明的实施例，所述VH包括SEQ ID NO：3、15、16或17所示的氨基酸序列，所述VL包括SEQ ID NO：4、18、19、20、21、22、23、24、25或26所示的氨基酸序列。
更进一步，根据本发明的实施例，具有SEQ ID NO:27所示氨基酸序列的HCDR1、SEQ ID NO:28-29任一所示氨基酸序列的HCDR2、SEQ ID NO:30所示氨基酸序列的HCDR3，且具有SEQ ID NO:31所示氨基酸序列的LCDR1、SEQ ID NO:32所示氨基酸序列的LCDR2、 SEQ ID NO:33所示氨基酸序列的LCDR3；或
具有SEQ ID NO:34所示氨基酸序列的HCDR1、SEQ ID NO:35所示氨基酸序列的HCDR2、SEQ ID NO:36所示氨基酸序列的HCDR3；且具有SEQ ID NO:37所示氨基酸序列的LCDR1、SEQ ID NO:38所示氨基酸序列的LCDR2、SEQ ID NO:39-40任一所示氨基酸序列的LCDR3。
进一步，根据本发明的实施例，具有选自SEQ ID NO:1、5-10中任一氨基酸序列所示重链可变区，选自SEQ ID NO:2、11-14中任一氨基酸序列所示轻链可变区；或
具有选自SEQ ID NO:3、15-17中任一氨基酸序列所示重链可变区，选自SEQ ID NO:4、18-26中任一氨基酸序列所示轻链可变区。
进一步，根据本发明的实施例，该抗人IL-36R抗体或其片段能够阻断IL36α、IL36β、和/或IL36γ诱导的IL36R+细胞炎性细胞因子释放，不具有效应子活性或效应子活性显著降低，其中，所述炎性细胞因子包括IL-8，所述效应子活性包括ADCC和CDC。
更进一步，根据本发明的实施例，所述抗体包括鼠源抗体、兔源抗体、人源抗体、嵌合抗体等；所述抗体部分包括Fab、Fab’、F(ab’)2、Fv、scFv、dAb等。
第二方面，本发明提供一种人源化抗人IL-36R抗体或其片段。根据本发明的实施例，该人源化抗人IL-36R抗体或其片段是在本发明前述抗人IL-36R抗体或其片段的基础上进行CDRs移植获得。
进一步，根据本发明的实施例，该人源化抗人IL-36R抗体或其片段具有IgG1亚类的重链恒定区，且在所述IgG1亚类重链恒定区的234和235位的亮氨酸(L)均突变为丙氨酸(A)。
第三方面，本发明提供一种多核苷酸。根据本发明的实施例，该多核苷酸编码本发明前述任一项所述抗人IL-36R抗体或其片段、或本发明前述任一项所述人源化抗人IL-36R抗体或其片段。
第四方面，本发明提供一种构建体，根据本发明的实施例，该构建体包含前述多核苷酸。
第五方面，本发明提供一种宿主细胞，根据本发明的实施例，该宿主细胞包含前文所述的多核苷酸或前文所述的核酸构建体。
第六方面，本发明提供一种组合物，根据本发明的实施例，该组合物包含本发明前述任一项所述抗人IL-36R抗体或其片段、前述任一项所述人源化抗人IL-36R抗体或其片段、前述多核苷酸和/或前述核酸构建体，以及可选的药学上可接受的辅料。
第七方面，本发明提供一种组合物。根据本发明的实施例，该组合物包含两种或两种以上抗人IL-36R单克隆抗体，所述两种或两种以上抗人IL-36R单克隆抗体选自前述任一项所述抗人IL-36R抗体或其片段、前述任一项所述人源化抗人IL-36R抗体或其片段；并且所述两种或两种以上抗人IL-36R单克隆抗体相互之间对人IL-36R无竞争结合关系。
第八方面，本发明提供一种制备抗人IL-36R抗体或其片段的方法。根据本发明的实施例，该方法包括：
(1)在合适的条件下，培养本发明前述的宿主细胞；
(2)分离回收抗人IL-36R抗体或其片段、人源化抗人IL-36R抗体或其片段。
第九方面，本发明提供前述任一项所述抗人IL-36R抗体或其片段、前述任一项所述人源化抗人IL-36R抗体或其片段、前述多核苷酸、前述构建体、前述宿主细胞和前述任一项所述组合物在制备治疗炎性疾病的药物中的应用。
进一步，根据本发明的实施例，所述炎性疾病是IL36相关的炎性疾病，所述药物通过阻断IL36α、IL36β、和/或IL36γ诱导与细胞受体IL36R的结合发挥治疗炎性疾病的作用。
更进一步，根据本发明的实施例，所述炎性疾病包括皮炎、银屑病、炎性肠病、关节炎、系统性红斑狼疮、炎性肺病、慢性肾病；其中银屑病包括脓疱型银屑病、泛发性银屑病和掌跖脓疱病。
进一步，根据本发明的实施例，所述炎性疾病具有皮肤损伤症状，所述药物通过阻断IL36α、IL36β、和/或IL36γ诱导与细胞受体IL36R的结合改善皮肤损伤。
第十方面，本发明提供了治疗患有癌症或炎性疾病的对象的方法。根据本发明的实施例，所述方法包括向所述对象施用治疗有效量的组合物，所述组合物为前述的组合物。
根据本发明的实施例，所述炎性疾病包括皮炎、银屑病、炎性肠病、关节炎、系统性红斑狼疮、炎性肺病和慢性肾病。
根据本发明的实施例，所述银屑病包括脓疱型银屑病、泛发性银屑病和掌跖脓疱病。
根据本发明的实施例，所述癌症为乳腺癌。
第十一方面，本发明提供了一种用于治疗或预防有需要的个体的IL-36R介导的疾病的方法。根据本发明的实施例，该方法包含任选地与另一种治疗剂结合施用治疗有效量的前述的组合物。
为更好理解本发明，首先定义一些术语。其他定义则贯穿具体实施方式部分而列出。
术语“IL-36R”，是细胞表面能与IL-36相互作用的膜蛋白，能够与IL-36α、IL-36β、IL-36γ、以及IL-36Ra结合。其中IL-36α、IL-36β、IL-36γ为IL-36R受体激动剂，IL-36Ra为IL-36R受体拮抗剂。在人体内，编码IL-36Ra的基因失活突变会导致IL-36R信号失调，主要表现为弥漫型脓疱银屑病，这表明IL-36在银屑病的皮肤炎症中有重要作用。研究表明IL-36在其他器官的炎症性疾病中也有重要作用。
术语“特异性结合”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。术语“免疫结合”是指发生在抗体分子和抗原(对于该抗原而言抗体为特异性的)之间的特异性结合反应。免疫结合相互作用的强度或亲和力可以相互作用的平衡解离常数(KD)表示，其中KD值越小，表示亲和力越高。两分子间的的免疫结合性质可使用本领域中公知的方法定量。一种方法涉及测量抗原结合位点/抗原复合物形成和解离的速度。指特定抗体-抗原相互作用的“结合速率常数”(Ka或Kon)和“解离速率常数”(Kd或Koff)两者都可通过浓度及缔合和解离的实际速率而计算得出，参见Malmqvist M,1993,Nature,361:186-187。Kd/Ka的比率等于解离常数KD，参见Davies DR等,1990,Annual  Rev Biochem.,59:439-473。可用任何有效的方法测量KD、Ka和Kd值。在优选的实施方案中，用生物发光干涉测量法来测量解离常数。在其它优选的实施方案中，可用表面等离子共振技术(例如Biacore)或KinExa来测量解离常数。
本文中的术语“抗体”意在包括全长抗体及其任何抗原结合片段(即，抗原结合部分)或单链。全长抗体是包含至少两条重(H)链和两条轻(L)链的糖蛋白，重链和轻链由二硫键连接。各重链由重链可变区(简称VH)和重链恒定区构成。重链恒定区由三个结构域构成，即CH1、CH2和CH3。各轻链由轻链可变区(简称VL)和轻链恒定区构成。轻链恒定区由一个结构域CL构成。VH和VL区还可以划分为称作互补决定区(CDR)的高变区，其由较为保守的框架区(FR)区分隔开。各VH和VL由三个CDR以及四个FR构成，从氨基端到羧基端以FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4的顺序排布。重链和轻链的可变区包含与抗原相互作用的结合域。抗体的恒定区可以介导免疫球蛋白与宿主组织或因子的结合，包括多种免疫系统细胞(例如，效应细胞)和传统补体系统的第一组分(C1q)。
术语“单克隆抗体”或“单抗”或“单克隆抗体组成”是指单一分子组成的抗体分子制品。单克隆抗体组成呈现出对于特定表位的单一结合特异性和亲和力。
本文中的术语，抗体的“抗原结合片段”(或简称为抗体片段)，是指抗体的保持有特异结合抗原能力的一个或多个片段。已证实，抗体的抗原结合功能可以通过全长抗体的片段来实施。包含在抗体的“抗原结合部分”中的结合片段的例子包括(i)Fab片段，由VL、VH、CL和CH1构成的单价片段；(ii)F(ab′)2片段，包含铰链区二硫桥连接的两个Fab片段的二价片段；(iii)由VH和CH1构成的Fd片段；(iv)由抗体单臂VL和VH构成的Fv片段；(v)由VH构成的dAb片段(Ward et al.，(1989)Nature 341：544-546)；(vi)分离的互补决定区(CDR)；以及(vii)纳米抗体，一种包含单可变结构域和两个恒定结构域的重链可变区。此外，尽管Fv片段的两个结构域VL和VH由不同的基因编码，它们可以通过重组法经由使两者成为单蛋白链的合成接头而连接，其中VL和VH区配对形成单价分子(称为单链Fc(scFv)；参见例如Bird et al.，(1988)Science 242：423-426；and Huston et al.，(1988)Proc.Natl.Acad.Sci.USA 85：5879-5883)。这些单链抗体也意在包括在术语涵义中。这些抗体片段可以通过本领域技术人员已知的常用技术而得到，且片段可以通过与完整抗体相同的方式进行功能筛选。
本发明的抗原结合片段包括能够特异性结合抗原分子的那些。抗体结合片段的实例包括例如但不限于Fab、Fab'、F(ab') 2、Fv片段、单链Fv(scFv)片段和单结构域片段。
Fab片段含有轻链的恒定结构域和重链的第一恒定结构域(CH1)。Fab'片段与Fab片段的不同之处在于在重链CH1结构域的羧基末端处的少数残基的添加，包括来自抗体铰链区的一个或多个半胱氨酸。通过切割在F(ab')2胃蛋白酶消化产物的铰链半胱氨酸处的二硫键产生Fab'片段。抗体片段的另外化学偶联是本领域普通技术人员已知的。Fab和F(ab')2片段缺乏完整抗体的片段可结晶(Fc)区，从动物的循环中更快速地清除，并且可能具有比完整抗体更少的非特异性组织结合(参见例如，Wahl等人，1983，J.Nucl.Med.24:316)。
如本领域通常理解的，“Fc”区是不包含抗原特异性结合区的抗体的片段可结晶恒定区。在IgG、IgA和IgD抗体同种型中，Fc区由两个相同的蛋白质片段组成，衍生自抗体的两条 重链的第二和第三恒定结构域(分别为CH2和CH3结构域)。IgM和IgE Fc区在每条多肽链中含有三个重链恒定结构域(CH2、CH3和CH4结构域)。
术语“人源化抗体”，人源化抗体主要指鼠源单克隆抗体以基因克隆及DNA重组技术改造，重新表达的抗体，其大部分氨基酸序列为人源序列取代，基本保留亲本鼠单克隆抗体的亲和力和特异性，又降低了其异源性。
术语“嵌合抗体”是指重链和/或轻链的一部分与衍生自特定物种或属于特定抗体类别或亚类的抗体中的相应序列相同或同源，而链的剩余部分与衍生自另一物种或属于另一抗体类别或亚类的抗体中的相应序列相同或同源，以及此类抗体的片段，只要它们展现出期望的生物学活性(美国专利No.4,816,567；Morrison SL等,Proc.Natl.Acad.Sci.USA,81:6851-6855,1984)。例如，术语“嵌合抗体”可包括这样的抗体(例如人鼠嵌合抗体)，其中抗体的重链和轻链可变区来自第一抗体(例如鼠源抗体)，而抗体的重链和轻链恒定区来自第二抗体(例如人抗体)。
术语“重链”，重链(heavy chain，H链)大小约为轻链的2倍，含450～550个氨基酸残基，分子量约为55或75kD。每条H链含有4～5个链内二硫键所组成的环肽。不同的H链由于氨基酸组成的排列顺序、二硫键的数目和位置、含的种类和数量不同，其抗原性也不相同，根据H链抗原性的差异可将其分为5类：μ链、γ链、α链、δ链和ε链，不同H链与L链(κ或λ链)组成完整免疫球蛋白的分子分别称之为IgM、IgG、IgA、IgD和IgE。γ、α和δ链上含有4个肽，μ和ε链含有5个环肽。
术语“轻链”，轻链(light chain，L)指在免疫球蛋白单体分子中相对于重链而论，在分子量上较小的多肽链。每条轻链近氨基末端(N端)1/2区域内的氨基酸组成序列多变处为轻链可变区(VL)，是Ig分子与抗原结合部位的一个组成部分。其余1/2区域内的氨基酸组成及排列顺序相对稳定处为轻链恒定区(CL)。由于轻链恒定区内氨基酸序列存在某些差异，故轻链有k和λ两型。
术语“种系”，又称胚系(germline)，抗体形成细胞具有编码Ig分子的全部基因(即有限数量的C基因和未知数量的V基因，它是通过长期进化形成并通过生殖细胞从亲代传给子代，人免疫球蛋白的重链基因由V-D-J-C基因片段编码而成；轻链基因由V-J-C基因片段编码而成，基因片段的个数不等。人重链基因定位于第14号染色体长臂，跨度约1,100kb，由V、D、J和C四种基因片段组成，包含约95个Va基因片段(分VHl～VH7七个家族，其中功能性基因片段65个、27个D基因片段、6个JH基因片段和9个CH基因片段。Va基因片段位于上游，D基因片段位于VH和JH基因簇之间，JH基因位于DH下游，与下游C基因区相隔7Kb左右，CH基因成簇(cluster)排列，跨度约200kb，P和S基因紧位于JH基因片段下游，C8下游依次是Cγ、Cα和Cε。人轻链基因分为λ和x基因，分别定位于第22号染色体长臂和第2号染色体短臂。功能性VK基因片段约40个，Vc基因片段后是5个功能性J和1个Cκ；Vλ基因片段约30个，4个Jλ基因片段和4个Cλ基因片段。
术语“载体”、“核酸构建体”是指能够运输与其连接的另一种核酸的核酸分子。一种类型的载体是“质粒”，其是指其中可以连接另外的DNA区段的环状双链DNA环。另一种 类型的载体是病毒载体，其中额外的DNA区段可以连接到病毒基因组中。某些载体能够在它们被导入的宿主细胞中自主复制(例如，具有细菌复制起点和游离型哺乳动物载体的细菌载体)。其他载体(例如非附加型哺乳动物载体)可以在导入宿主细胞后整合到宿主细胞的基因组中，并由此与宿主基因组一起复制。此外，某些载体能够指导它们有效连接的基因的表达。这种载体在本文中被称为“重组表达载体”(或简称为“表达载体”)。通常，在重组DNA技术中有用的表达载体通常以质粒的形式存在。然而，也包括其他形式的表达载体，如病毒载体(例如，复制缺陷型逆转录病毒，腺病毒和腺伴随病毒)，其起到等同的功能。
术语“多核苷酸”旨在包括DNA分子和RNA分子。多核苷酸可以是单链或双链的，并且可以是cDNA或其片段。
术语“多肽”是指包含至少两个连续连接的氨基酸残基的链，对链的长度没有上限。蛋白质中的一个或多个氨基酸残基可含有修饰，例如但不限于糖基化，磷酸化或二硫键。“蛋白质”可以包含一种或多种多肽。
术语“宿主细胞”在其中载体可以增殖并且其DNA可以表达的细胞，所述细胞可以是原核细胞或者真核细胞。该术语还包括受试宿主细胞的任何后代。应理解，并不是所有的后代都与亲本细胞相同，因为在复制过程中可能会发生突变，这类后代被包括在内。
本发明实施例的抗人IL-36R抗体或其片段至少具有以下有益的技术效果之一：
1、本发明实施例的抗人IL-36R抗体不仅对人IL36R具有较高的亲和力，而且在Jurkat-hIL36R-IL1Racp-NF-κB报告基因细胞模型、表皮鳞癌A431细胞中均能够抑制IL-36诱导的炎性细胞因子分泌，因此在治疗炎性疾病特别是银屑病等炎性相关的皮肤疾病时预期具有较好的治疗效果。
2、本发明实施例的抗人IL-36R抗体或其片段对癌症和炎症疾病具有良好的治疗效果，并且，该抗体或其片段兼顾了动物模型的适应性和动物试验的有效性，在杂交瘤筛选过程中即选择了对人IL-36R、猴IL-36R均具有特异性结合能力的候选抗体，在人源化改造过程中也兼顾了对猴IL-36R的特异性，因此本发明的人源化抗人IL-36R抗体可以直接施用于猴皮肤炎症动物模型用于临床期评估治疗效果。
3、发明人研究发现抗人IL-36R抗体能结合到靶细胞膜表面IL-36R激发效应因子活性，通过ADCC或CDC效应杀伤靶细胞，从而释放炎症细胞因子、加重局部炎症和损伤。IgG4亚型抗体效应子活性低但稳定性较差，本发明的抗人IL-36R抗体采用IgG1亚型的重链恒定区并对其进行突变改造，即保持了IgG1亚型抗体的稳定性又消除或降低了效应子活性。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1：本发明实施例的鼠源单克隆抗体与hu-IL36R蛋白的结合活性的结果示意图；
图2：本发明实施例的人-鼠嵌合抗体与重组HEK293细胞表面hu-IL36R和cyno-IL36R的结合活性的结果示意图，其中，A为人-鼠嵌合抗体与重组HEK293细胞表面hu-IL36R的结合曲线，B为人-鼠嵌合抗体与重组HEK293细胞表面cyno-IL36R的结合曲线；
图3：本发明实施例的Jurkat-hIL36R-IL1Racp-NF-κB细胞模型中人-鼠嵌合抗体对IL-36诱导的炎性细胞因子分泌的抑制活性的结果示意图；
图4：本发明实施例的表皮鳞癌A431细胞中人-鼠嵌合抗体对IL-36诱导的炎性细胞因子分泌的抑制活性的结果示意图，其中，A为在A431细胞中嵌合抗体对IL-36α诱导的IL-8分泌的抑制活性的结果示意图，B为在A431细胞中嵌合抗体对IL-36β诱导的IL-8分泌的抑制活性的结果示意图，C为在A431细胞中嵌合抗体对IL-36γ诱导的IL-8分泌的抑制活性的结果示意图，
图5：本发明实施例的人源化抗体与人IL36R和食蟹猴IL36R的结合的结果示意图，其中，A为人源化抗体与人IL36R的结合的结果示意图，B为人源化抗体与食蟹猴IL36R的结合的结果示意图；
图6：本发明实施例的表皮鳞癌A431细胞中人源化抗体对IL-36诱导的炎性细胞因子分泌的抑制活性的结果示意图，其中，A为在A431细胞中人源化抗体对IL-36α诱导的IL-8分泌的抑制活性的结果示意图，B为在A431细胞中人源化抗体对IL-36β诱导的IL-8分泌的抑制活性的结果示意图，C为在A431细胞中人源化抗体对IL-36γ诱导的IL-8分泌的抑制活性的结果示意图。
发明详细描述
下面详细描述本发明的实施例，所述实施例的示例在附图中示出，其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。
需要说明的是，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。进一步地，在本发明的描述中，除非另有说明，“多个”的含义是两个或两个以上。
实施例1：抗人IL-36R单克隆抗体杂交瘤细胞制备与筛选
1.1杂交瘤制备和筛选
将得到的人白介素36受体蛋白(带His标签，货号CI17，近岸蛋白质科技有限公司)按常规方法(Lonberg,N.,et al.,nature 368(1994)856-859；Fishwild,D.M.,et al.,Nat.Biotechnol.14(1996)845-851and WO 98/24884)免疫10只Balb/c小鼠。每只小鼠初次免疫蛋白用量为50ug，此后进行3次加强免疫，蛋白用量为25ug/小鼠。
利用抗原特异性酶联免疫吸附法(ELISA)来测定小鼠血清中的IL-36R效价：将浓度为1ug/ml的白介素36受体蛋白(IL-36R，带Fc标签，货号CJ62，近岸蛋白质科技公司) 包被于96孔板中，每孔100μl，4℃孵育过夜，然后洗去包被液，每孔添加200ul封闭液(protein-free blocking buffer)，室温静置2小时。将小鼠血清在PBSA(含1％BSA的PBS溶液)中预先稀释200倍，并以1:2连续稀释12个梯度至409600倍。洗去封闭液后，将稀释的小鼠血清加入96孔板，室温孵育1小时，用PBST洗涤96孔，然后每孔加入100ul以1:3000稀释的二抗(HRP标记的羊抗鼠IgG)，室温静置45分钟，PBST洗涤5遍，每孔加入50ul室温平衡的TMB，室温孵育10分钟，在450nm测量吸光度。
取血清效价较高的小鼠，取脾脏，分离得到小鼠B细胞，与预先制好备的小鼠骨髓瘤细胞P3X63Ag8.653以1:2的比例混合，进行电击融合；将融合后的杂交瘤细胞接种于384孔板中，加入适当培养基进行培养，经过7-10天，取杂交瘤细胞培养上清，用上述酶联免疫吸附法(ELISA)筛选阳性克隆。筛选得到若干强反应阳性克隆，通过有限稀释法将筛到的这些强反应阳性克隆单细胞华，最终得到的每个杂交瘤细胞只分泌同一种小鼠抗体，获得23株分泌鼠源抗人IL-36R的单克隆抗体，其对hu-IL36R蛋白的结合活性如图1所示。
1.2鼠源抗人IL-36R单克隆抗体序列的确定
将上述得到的分泌抗人IL-36R抗体杂交瘤细胞扩大培养后，按照RNAfast200试剂盒(上海飞捷生物技术有限公司)说明书步骤提取细胞总RNA；利用5×PrimeScript RT Master Mix(Takara)将杂交瘤细胞总RNA反转录成cDNA；使用简并引物(Anke Krebber.1997)和Extaq PCR试剂(Takara)扩增抗体轻链可变区IgVL(κ)和重链可变区VH序列；利用PCR clean-up Gel extraction试剂盒(Macherey-Nagel公司)纯化PCR扩增产物；按照pClone007Simple Vector Kit试剂盒(擎科生物科技有限公司)说明书将扩增PCR产物连接至T载体并转化大肠杆菌感受态细胞，菌株扩增、抽提质粒后进行DNA测序获得单克隆抗体可变区序列。测序结果如表1所示，表1a为重链可变区测序结果，表1b为轻链可变区测序结果。
表1a.鼠源抗人IL-36R单克隆抗体重链可变区氨基酸序列
Figure PCTCN2022075460-appb-000001
Figure PCTCN2022075460-appb-000002
Figure PCTCN2022075460-appb-000003
Figure PCTCN2022075460-appb-000004
表1b.鼠源抗人IL-36R单克隆抗体轻链可变区氨基酸序列
Figure PCTCN2022075460-appb-000005
Figure PCTCN2022075460-appb-000006
Figure PCTCN2022075460-appb-000007
Figure PCTCN2022075460-appb-000008
实施例2：抗人IL-36R嵌合抗体的制备与纯化
将上述鼠源抗人IL-36R单克隆抗体的重链可变区和公开发表的人单克隆抗体IgG1亚类的重链恒定区(具有两个替代突变Leu234Ala以及Leu235Ala，借此降低效应子功能如FcRn及补体结合而消除ADCC和CDC活性)拼接在一起，形成嵌合抗体的重链(hIgG1-LALA)，将该重链构建到哺乳动物细胞表达载体中；将鼠源抗人IL-36R单克隆抗体的轻链可变区和公开发表的人单克隆抗体Kappa亚类轻链恒定区拼接在一起，形成嵌合抗体的轻链，将该轻链构建到哺乳动物细胞表达载体中。
将构建好的抗人IL-36R嵌合抗体的重链质粒和轻链质粒配对混合，使用聚乙烯亚胺(PEI)转染HEK293细胞，约7天后收集细胞上清，使用Protein A纯化得到抗人IL-36R嵌合抗体蛋白，检测嵌合抗体蛋白的浓度、体积和蛋白总量，结果如表2所示。
表2.纯化获得的重组嵌合抗体
Figure PCTCN2022075460-appb-000009
Figure PCTCN2022075460-appb-000010
实施例3：抗人IL-36R嵌合抗体的活性和理化特征
3.1抗人IL-36R嵌合抗体对不同种属IL-36R的特异性和交叉反应性
分别将编码人IL-36R(huIL-36R,NCBI accession:NM_001351446)、小鼠IL-36R(muIL-36R,NCBI accession:NM_001356478)和食蟹猴IL-36R(cyno-IL36R,NCBI accession:XP_014968513)全长基因的质粒用聚乙烯亚胺(PEI)转染HEK293细胞，并挑选稳定高表达的单细胞备用。
将上述得到的表达人IL-36R的HEK293细胞以30，000/孔铺于96孔板中，随后每孔添加50ul上述得到的嵌合抗体，添加的嵌合抗体从100nM起始，并以1:3连续稀释8个梯度，随后在室温下静置1小时。用PBSA(含有1％BSA的PBS溶液)洗涤，加入50ul1:400稀释的二抗(FITC-羊抗人IgG)，4℃静置30分钟，随后用PBSA洗涤，最后用50ul PBSA重悬细胞，用流式细胞仪分析目标细胞群的平均荧光强度。
同样将上述表达小鼠IL-36R和食蟹猴IL-36R的HEK293细胞铺于96孔板中，随后按照上述流程检测嵌合抗体与这两个细胞的结合。结果如图2、表3(表3a.与HEK293细胞表面人IL-36R结合的嵌合抗体EC50值，表3b.与HEK293细胞表面猴IL-36R结合的嵌合抗体EC50值)所示，上述结果表明，15株嵌合抗体能以高亲和力结合重组HEK293细胞表面的huIL-36R，只有4株嵌合抗体能以高亲和力结合重组HEK293细胞表面的cyno-IL36R，所有的嵌合抗体都不能特异性结合重组HEK293细胞表面的muIL-36R。
表3a.与HEK293细胞表面人IL-36R结合的嵌合抗体EC50值
Figure PCTCN2022075460-appb-000011
Figure PCTCN2022075460-appb-000012
表3b.与HEK293细胞表面猴IL-36R结合的嵌合抗体EC50值
Figure PCTCN2022075460-appb-000013
3.2抗人IL-36R嵌合抗体与人IL-36R结合的动力学检测
利用Fortebio(BLITZ pro1.1.0.28)仪器分析嵌合抗与抗原huIL-36R的结合动力学参数。测定前先将AHC生物探针浸泡于PBS中10分钟；然后将该探针置于含100nM的抗体的PBS中300秒，捕获带hIgG1-Fc标签的抗体；进一步将探针与100nM抗原进行结合反应，结合时间400秒；之后将探针转移至PBS中，进行解离反应，时间为600秒。实验完毕，扣除空白亲本响应值，用软件进行1：1Langmuir结合模式拟合，计算抗原抗体结合的动力学常数，结果如表4所示。同样利用Fortebio(BLITZ pro1.1.0.28)仪器分析嵌合抗体与人IL-1R1的结合动力学参数，结合信号显示为0(结果未显示)。这表明所有的嵌合抗体特异性的结合人IL-36R。因此，表4的结果表明纯化得到的嵌合抗体在体外能以高亲和力特异性结合人IL-36R。
表4：嵌合抗体与huIL-36R结合的动力学检测
Figure PCTCN2022075460-appb-000014
Figure PCTCN2022075460-appb-000015
3.3抗人IL-36R嵌合抗体的表位竞争分析
使用Fortebio(BLITZ pro1.1.0.28)仪器测定嵌合抗体与人IL-36R的交叉竞争性结合。在每个测试中，利用NTA芯片捕获人IL-36R(带有His标签)，然后用PBSA(含有1％BSA的PBS溶液)来阻断未被利用的捕获位点，随后加入饱和量的嵌合抗体1，然后再加入嵌合抗体2以确定嵌合抗体2是否竞争性结合人IL-36R。如果这两个嵌合抗体结合人IL-36R的同一或近似表位，则不会观察到二级结合，如果嵌合抗体2结合了人IL-36R的不同结合位点，嵌合抗体2将与嵌合抗体1/人IL-36R复合物结合。
随后变换嵌合抗体1和2的上样顺序(之前先使用嵌合抗体1，此次则先使用嵌合抗体2)，再次测定这两个嵌合抗体的交叉竞争性结合。结果如表5所示，表5的结果表明纯化得到的不同嵌合抗体能结合人IL36R的不同表位。
表5：嵌合抗体与人IL-36R的交叉竞争反应
Figure PCTCN2022075460-appb-000016
3.4抗人IL-36R嵌合抗体的理化性质检测
分别对上述获得的嵌合抗体进行理化性质检测，包括单体率(SEC)、疏水性(HIC)、热稳定性(TM)及分子量测定(MALS)。
抗体单体率分析：将50ul浓度为1mg/ml的抗体进样至预平衡好的层析柱(TSKgel G3000SWXL 7.8*300(TOSHI)内，室温，0.7ml/min的流速流动35min，并同时检测机器(WATERS UPLC CLASS ACQUITYH)A280的吸收值，进入根据出峰时间和出峰体积来判断抗体的单体含量和比例。
抗体疏水性质分析：使用WATERS ARC仪器和疏水层析柱(TSKgel Butyl-NPR(4.6mmX3.5cm，Cat No 14947)使用说明，来分析抗体的疏水性质。
抗体热稳定性分析：使用Bio-rad的荧光定量PCR仪和ProteoSTAT Thermal shift assay kit(Cat No.ENZ-51027-K400)分析抗体的热稳定性(TM)。
嵌合抗体单体率(SEC)、疏水性(HIC)、热稳定性(TM)及分子量测定(MALS)的检测结果如表6所示，表明选定的6株嵌合抗体理化性质(包括不同pH条件下的热稳定性、单体率和疏水性)比较稳定可靠。
表6.嵌合抗体的初步理化性质分析
Figure PCTCN2022075460-appb-000017
3.5抗人IL-36R嵌合抗体对重组报告基因细胞株的作用
将编码人IL-36R(huIL-36R,NCBI accession:NM_001351446)、编码人IL-1RAcP以及编码Luc2P-NFkB基因的3个质粒依次稳定转染至Jurkat细胞(上海细胞库,Cat.No.TCHU123)，挑选稳定高表达的单细胞备用，建立Jurkat-hIL36R-IL1Racp-NF-κB报告基因细胞株。
将上述所得的嵌合抗体配制成167nM、10nM和0.7nM三个浓度，分别与近似EC50浓度的IL-36R配体混合(之前已预先测定了IL36α、IL36β和IL36γ这三个配体各自刺激报告基因细胞产生荧光信号的EC50浓度)；混合后的溶液按照50ul/孔加入到白色底透96孔板中。将上述所得的报告基因细胞离心换液，然后按1x10 5个细胞/孔加入到上述白色底透96孔板中，过夜培养后每孔加入100ul Bio-Lite显色剂，读取化学发光值。结果如图3所示，该结果表明嵌合抗体能以剂量依赖的方式抑制经IL-36R配体(IL36α、IL36β和IL36γ)刺激Jurkat细胞分泌炎性细胞因子，而同型抗体对照则不能。
3.6抗人IL-36R嵌合抗体在表皮鳞癌A431细胞中抑制IL-36诱导的IL-8分泌
A431细胞在IL36R配体(IL36α、IL36β和IL36γ)的刺激下会产生细胞因子IL-8，而抗IL-36R抗体则会抑制A431细胞产生IL-8.
将上述所得的嵌合抗体从30ug/ml起始按照3倍倍比稀释10个梯度，分别与近似EC50浓度的IL-36R配体混合(之前已预先测定了IL36α、IL36β和IL36γ这三个配体各自的EC50浓度)；混合后的溶液按照50ul/孔加入到白色底透96孔板中。将A431细胞离心换液，然后按1x10 5个细胞/孔加入到上述白色底透96孔板中，培养48小时，然后使用R&D Systems DUO-SET ELISA试剂盒，根据制造商提供的标准方案，通过ELISA评估细胞上清液中IL-8的水平。结果如图4所示，上述结果证实嵌合抗体能以剂量依赖的方式抑制经IL-36R配体(IL36α、IL36β和IL36γ)刺激的A431细胞分泌炎性细胞因子IL-8。
实施例4：抗人IL-36R人源化抗体的设计与制备
4.1抗人IL-36R人源化抗体的设计和工程化改造
为降低小鼠抗体潜在的免疫原性，需将先前得到的小鼠抗人IL-36R抗体人类化，即将小鼠抗体中的鼠源序列尽可能多的变更为人源序列，同时在此过程中，鼠源抗体的结合特异性、动力学参数、理化性质、稳定性等特性应予以保留。为达此目的，基于框架同源性、CDR结构、保守标准残基、保守接口封装残基和其他参数选择小鼠前导序列的人类框架序列。所形成的人源化抗体，包括结合特异性、结合动力学参数、理化性质等，选择与亲代鼠源抗体相比显示较好或相等结合的人源化抗体进行后续进一步验证。在此，选择鼠源抗体A10-6B12和A19-35D19进行人源化(克隆至人类IgG1-LALA/κ骨架中，可变区序列见表7)。
表7A.A10-6B12人源化抗体的可变区及其CDR氨基酸序列
Figure PCTCN2022075460-appb-000018
Figure PCTCN2022075460-appb-000019
注：CDRs区以下划线标出。
表7B.A10-6B12人源化抗体的轻重链配对设计
Figure PCTCN2022075460-appb-000020
表7C.A19-35D19人源化抗体的可变区及其CDR氨基酸序列
Figure PCTCN2022075460-appb-000021
Figure PCTCN2022075460-appb-000022
Figure PCTCN2022075460-appb-000023
注：CDRs区以下划线标出。
表7D.A19-35D19人源化抗体的轻重链配对设计
Figure PCTCN2022075460-appb-000024
4.2抗人IL-36R人源化抗体的制备和纯化
将上述人源化抗人IL-36R单克隆抗体的重链可变区和公开发表的人单克隆抗体IgG1亚类的重链恒定区(具有两个替代突变Leu234Ala以及Leu235Ala，借此降低效应子功能如FcRn及补体结合而消除ADCC和CDC活性)拼接在一起，形成人源化抗体的重链(hIgG1-LALA)，将该重链构建到哺乳动物细胞表达载体中；同时也将上述人源化抗IL-36R单克隆抗体的重链可变区和公开发表的人单克隆抗体IgG1亚类的重链恒定区1(hIgG1-VH1)拼接在一起，形成人源化抗体的重链Fd(VH-CH1)，将该重链Fd同样构建到哺乳动物细胞表达载体中。
将人源化抗人IL-36R单克隆抗体的轻链可变区和公开发表的人单克隆抗体Kappa亚类轻链恒定区拼接在一起，形成人源化抗体的轻链，将该轻链构建到哺乳动物细胞表达载体中。
将构建好的抗人IL-36R嵌合抗体的重链质粒(包括重链Fd质粒)和轻链质粒配对混合，使用聚乙烯亚胺(PEI)转染HEK293细胞，约7天后收集细胞上清，使用Protein A纯化得到抗人IL-36R嵌合抗体蛋白(该人源化抗体为hIgG1-LALA亚型，同时也得到部分人 源化抗体hIgG1-LALA亚型的Fab形式，见表8)。
表8：纯化得到的抗人IL-36R人源化抗体
Figure PCTCN2022075460-appb-000025
实施例5：抗人IL-36R人源化抗体的活性和功能
5.1人源化抗体的亲和力参数测定
利用Fortebio(BLITZ pro1.1.0.28)仪器分析人源化抗与抗原huIL-36R的结合动力学参数。测定前先将AHC生物探针浸泡于PBS中10分钟；然后将该探针置于含100nM的抗体的PBS中300秒，捕获带hIgG1-Fc标签的抗体；进一步将探针与100nM抗原进行结合反应，结合时间400秒；之后将探针转移至PBS中，进行解离反应，时间为600秒。实验完毕，扣除空白亲本响应值，用软件进行1：1Langmuir结合模式拟合，计算抗原抗体结合的动力学常数。
同样使用Fortebio(BLITZ pro1.1.0.28)仪器测定抗体(Fabric形式)与人IL-36R的结合活性。在每个测试中，利用NTA芯片捕获人IL-36R(带有His标签)，然后用PBSA(含有1％BSA的PBS溶液)来阻断未被利用的捕获位点，进一步将探针与100nM抗体Fab进行结合反应，结合时间400秒；之后将探针转移至PBS中，进行解离反应，时间为600秒。 实验完毕，扣除空白亲本响应值，用软件进行1：1Langmuir结合模式拟合，计算抗原抗体结合的动力学常数。上述人源化抗体和Fab抗体的亲和力检测结果如表9所示。
同样利用Fortebio(BLITZ pro1.1.0.28)仪器分析嵌合抗体与人IL-1R1的结合动力学参数，结合信号显示为0(结果未显示)，这表明所有的人源化抗体特异性的结合人IL-36R。
表9：人源化抗体与人IL-36R结合的动力学参数
Figure PCTCN2022075460-appb-000026
表9的结果表明，A10-6B12和A19-35D19经过人源化改造之后，大多数人源化抗体都保持了与人IL36R的高亲和力结合。
5.2抗人IL-36R人源化抗体对不同种属IL-36R的交叉反应性
将上述得到的表达人IL-36R的HEK293细胞以30，000/孔铺于96孔板中，随后每孔添加50ul上述得到的人源化抗体，添加的人源化抗体从100nM起始，并以1:3连续稀释8个梯度，随后在室温下静置1小时。用PBSA(含有1％BSA的PBS溶液)洗涤，加入50ul1:400稀释的二抗(FITC-羊抗人IgG)，4℃静置30分钟，随后用PBSA洗涤，最后用50ul PBSA重悬细胞，用流式细胞仪分析目标细胞群的平均荧光强度。同样将上述表达小鼠IL-36R和食蟹猴IL-36R的HEK293细胞铺于96孔板中，随后按照上述流程检测人源化抗体与这两个细胞的结合。
结果如图5、表10所示，上述结果表明大多人源化抗体都能以高亲和力结合细胞表面表达的huIL-36R和cyno-IL36R。所有的人源化抗体都不能结合细胞表面的muIL-36R。
表10.人源化抗体与人IL36R和食蟹猴IL36R的结合活性
Figure PCTCN2022075460-appb-000027
5.3抗人IL-36R人源化抗体在A431细胞中抑制IL-36诱导的IL-8分泌
A431细胞在IL36R配体(IL36α、IL36β和IL36γ)的刺激下会产生炎症细胞因子IL-8而IL-36R抗体则会抑制A431细胞产生IL-8.
将上述所得的人源化抗体从30ug/ml起始按照3倍倍比稀释10个梯度，分别与近似EC50浓度的IL-36R配体混合(之前已预先测定了IL36α、IL36β和IL36γ这三个配体各自的EC50浓度)；混合后的溶液按照50ul/孔加入到白色底透96孔板中。将A431细胞离心换液，然后按1x10 5个细胞/孔加入到上述白色底透96孔板中，培养48小时，然后使用R&D Systems DUO-SET ELISA试剂盒，根据制造商提供的标准方案，通过ELISA评估细胞上清液中IL-8的水平。该结果(表11和图6)证实人源化抗体能以剂量依赖的方式高效抑制经IL-36R配体(IL36α、IL36β和IL36γ)刺激的A431细胞分泌炎性细胞因子IL-8。
表11.人源化抗体在体外阻断A431细胞经IL36刺激释放炎性细胞因子IL8
Figure PCTCN2022075460-appb-000028
综上所述，本发明实施例的人IL-36R抗体对人IL36R具有较高的亲和力，并且能抑制IL-36诱导的炎性细胞因子分泌，并且，该抗体对人和猴IL-36R均具有特异性结合能力。此外，本发明实施例的人IL-36R抗体采用IgG1亚型的重链恒定区并对其进行突变改造，即保持了IgG1亚型抗体的稳定性又消除或降低了效应子活性，避免了抗人IL-36R抗体结合到靶细胞膜表面IL-36R激发效应子活性，释放炎症细胞因子、加重局部炎症和损伤，使本发明实施例的人IL-36R抗体抗炎和抗肿瘤效果更好，安全性更高。
得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示意性实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不一定指的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。

Claims (28)

  1. 一种抗人IL-36R抗体或其片段，其特征在于，能够特异性结合人IL-36R和猴IL-36R，并且对人IL-36R的结合能力高于对猴IL-36R的结合能力。
  2. 根据权利要求1所述抗人IL-36R抗体或其片段，其特征在于，与人IL-36R结合的KD值<5×10 9。
  3. 根据权利要求1所述抗人IL-36R抗体或其片段，其特征在于，包括：
    重链可变区，所述重链可变区(VH)包括抗原决定区(CDRs)1、2和3，其中，VH CDR1包含与所选VH CDR1氨基酸序列具有至少75％同一性的氨基酸序列；VH CDR2包含与所选VH CDR2氨基酸序列具有至少75％同一性的氨基酸序列；VH CDR3与所选VH CDR3氨基酸序列具有至少75％同一性的氨基酸序列；
    轻链可变区，所述轻链可变区(VL)包括CDRs 1、2和3，其中，VL CDR1包含与所选VL CDR1氨基酸序列具有至少75％同一性的氨基酸序列；VL CDR2包含与所选VL CDR2氨基酸序列具有至少75％同一性的氨基酸序列；VL CDR3与所选VL CDR3氨基酸序列具有至少75％同一性的氨基酸序列；
    其中，所述所选VH CDRs 1、2和3的氨基酸序列以及所述所选VL CDRs 1、2和3的氨基酸序列选自下列之一：
    所述所选VH CDRs 1和3的氨基酸序列分别如SEQ ID NO：27和30所示，VH CDR2具有GEIFPNX 1GR所示的氨基酸序列，且所述所选VL CDRs 1、2和3的氨基酸序列如SEQ ID NO：31、32和33所示；
    所述所选VH CDRs 1、2和3的氨基酸序列分别如SEQ ID NO：34、35和36所示，且所述所选VL CDRs 1和2的氨基酸序列如SEQ ID NO：37和38所示，VL CDR3具有QQYX 2X 3YPLT所示的氨基酸序列，
    其中，
    X 1为S或T；
    X 2为N或T；
    X 3为S或T。
  4. 根据权利要求3所述抗人IL-36R抗体或其片段，其特征在于，所述VH包括与SEQ ID NO：5具有至少75％同一性氨基酸序列，且所述VL包括与SEQ ID NO：11具有至少75％同一性氨基酸序列。
  5. 根据权利要求3所述抗人IL-36R抗体或其片段，其特征在于，所述VH包括与SEQ ID NO：15具有至少75％同一性氨基酸序列，且所述VL包括与SEQ ID NO：18具有至少75％同一性氨基酸序列。
  6. 根据权利要求4所述抗人IL-36R抗体或其片段，其特征在于，所述VH包括SEQ ID NO:1、5、6、7、8、9或10所示的氨基酸序列，所述VL包括SEQ ID NO：2、11、12、13 或14所示的氨基酸序列。
  7. 根据权利要求5所述抗人IL-36R抗体或其片段，其特征在于，所述VH包括SEQ ID NO：3、15、16或17所示的氨基酸序列，所述VL包括SEQ ID NO：4、18、19、20、21、22、23、24、25或26所示的氨基酸序列。
  8. 根据权利要求1所述抗人IL-36R抗体或其片段，其特征在于，所述抗体或其片段能够阻断IL36α、IL36β、和/或IL36γ诱导的IL36R +细胞炎性细胞因子释放，不具有效应子活性或效应子活性显著降低，
    其中，所述炎性细胞因子包括IL-8，所述效应子活性包括ADCC和CDC。
  9. 根据权利要求1所述抗人IL-36R抗体或其片段，其特征在于，所述抗体或其片段为鼠源抗体或其抗原结合片段、兔源抗体或其抗原结合片段、人源抗体或其抗原结合片段，或嵌合抗体或其抗原结合片段。
  10. 根据权利要求1所述抗人IL-36R抗体或其片段，其特征在于，所述抗体或其片段为Fab、Fab’、F(ab’)2、Fv、scFv或dAb。
  11. 一种人源化抗人IL-36R抗体或其片段，其特征在于，是在权利要求1-10任一所述抗人IL-36R抗体或其片段的基础上进行CDRs移植获得的。
  12. 根据权利要求11所述的人源化抗人IL-36R抗体或其片段，其特征在于，所述人源化抗人IL-36R抗体或其片段具有IgG1亚类的重链恒定区，且在所述IgG1亚类重链恒定区的234和235位的亮氨酸(L)均突变为丙氨酸(A)。
  13. 一种多核苷酸，其编码权利要求1-10中任一项所述抗人IL-36R抗体或其片段、或权利要求11-12中任一项所述人源化抗人IL-36R抗体或其片段。
  14. 构建体，其特征在于，包含权利要求13所述的多核苷酸。
  15. 宿主细胞，其特征在于，包含权利要求11所述的多核苷酸或权利要求12所述的核酸构建体。
  16. 一种组合物，其特征在于，包含权利要求1-10中任一项所述抗人IL-36R抗体或其片段、权利要求11-12中任一项所述人源化抗人IL-36R抗体或其片段、权利要求13所述多核苷酸和/或权利要求14所述核酸构建体，以及可选的药学上可接受的辅料。
  17. 一种组合物，其特征在于，包含两种或两种以上抗人IL-36R单克隆抗体，所述两种或两种以上抗人IL-36R单克隆抗体选自权利要求1-10中任一项所述抗人IL-36R抗体或其片段或权利要求11-12中任一项所述人源化抗人IL-36R抗体或其片段；
    并且所述两种或两种以上抗人IL-36R单克隆抗体相互之间对人IL-36R无竞争结合关系。
  18. 一种制备抗人IL-36R抗体或其片段的方法，包括：
    (1)在合适的条件下，培养权利要求15所述的宿主细胞；
    (2)分离回收抗人IL-36R抗体或其片段、人源化抗人IL-36R抗体或其片段。
  19. 权利要求1-10中任一项所述抗人IL-36R抗体或其片段、权利要求11-12中任一项 所述人源化抗人IL-36R抗体或其片段、权利要求13所述多核苷酸、权利要求14所述构建体、权利要求15所述宿主细胞、权利要求16所述组合物和权利要求17所述组合物在制备治疗炎性疾病的药物中的应用。
  20. 根据权利要求19所述的应用，其特征在于，所述炎性疾病是IL36相关的炎性疾病，所述药物通过阻断IL36α、IL36β、和/或IL36γ诱导与细胞受体IL36R的结合发挥治疗炎性疾病的作用。
  21. 根据权利要求19所述的应用，其特征在于，所述炎性疾病包括皮炎、银屑病、炎性肠病、关节炎、系统性红斑狼疮、炎性肺病和慢性肾病。
  22. 根据权利要求21所述的应用，其特征在于，所述银屑病包括脓疱型银屑病、泛发性银屑病和掌跖脓疱病。
  23. 根据权利要求19所述的应用，其特征在于所述炎性疾病具有皮肤损伤症状，所述药物通过阻断IL36α、IL36β、和/或IL36γ诱导与细胞受体IL36R的结合改善皮肤损伤。
  24. 治疗患有癌症或炎性疾病的对象的方法，所述方法包括向所述对象施用治疗有效量的组合物，所述组合物为权利要求16或17所述的组合物。
  25. 根据权利要求24所述的方法，其特征在于，所述炎性疾病包括皮炎、银屑病、炎性肠病、关节炎、系统性红斑狼疮、炎性肺病和慢性肾病。
  26. 根据权利要求25所述的方法，其特征在于，所述银屑病包括脓疱型银屑病、泛发性银屑病和掌跖脓疱病。
  27. 根据权利要求24所述的方法，其特征在于，所述癌症为乳腺癌。
  28. 一种用于治疗或预防有需要的个体的IL-36R介导的疾病的方法，其包含任选地与另一种治疗剂结合施用治疗有效量的权利要求16或17所述的组合物。
PCT/CN2022/075460 2021-02-08 2022-02-08 抗人il-36r抗体及其应用 WO2022166977A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110183575.1 2021-02-08
CN202110183575 2021-02-08

Publications (1)

Publication Number Publication Date
WO2022166977A1 true WO2022166977A1 (zh) 2022-08-11

Family

ID=82742000

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/075460 WO2022166977A1 (zh) 2021-02-08 2022-02-08 抗人il-36r抗体及其应用

Country Status (2)

Country Link
CN (1) CN114907477A (zh)
WO (1) WO2022166977A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115724975A (zh) * 2022-10-20 2023-03-03 江苏荃信生物医药股份有限公司 抗人白介素36受体单克隆抗体及其应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130236471A1 (en) * 2011-11-16 2013-09-12 Boehringer Ingelheim Gmbh Anti il-36r antibodies
US20180273627A1 (en) * 2017-03-27 2018-09-27 Boehringer Ingelheim International Gmbh Anti il-36r antibodies combination therapy
WO2020018503A2 (en) * 2018-07-16 2020-01-23 Regeneron Pharmaceuticals, Inc. Anti-il36r antibodies
CN111500668A (zh) * 2020-06-29 2020-08-07 南京诺艾新生物技术有限公司 一种用于测定人IL-36/IL36R/IL1RAcP通路抑制剂的生物学活性的方法
US20200282053A1 (en) * 2019-03-08 2020-09-10 Boehringer Ingelheim International Gmbh Anti-il-36r antibody formulations
CN112094349A (zh) * 2020-11-04 2020-12-18 上海华奥泰生物药业股份有限公司 靶向于白介素36r的抗体及其制备方法和应用
CN112272674A (zh) * 2018-03-14 2021-01-26 勃林格殷格翰国际有限公司 抗-il-36r抗体用于治疗泛发性脓疱性银屑病的用途

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130236471A1 (en) * 2011-11-16 2013-09-12 Boehringer Ingelheim Gmbh Anti il-36r antibodies
US20180273627A1 (en) * 2017-03-27 2018-09-27 Boehringer Ingelheim International Gmbh Anti il-36r antibodies combination therapy
CN112272674A (zh) * 2018-03-14 2021-01-26 勃林格殷格翰国际有限公司 抗-il-36r抗体用于治疗泛发性脓疱性银屑病的用途
WO2020018503A2 (en) * 2018-07-16 2020-01-23 Regeneron Pharmaceuticals, Inc. Anti-il36r antibodies
US20200282053A1 (en) * 2019-03-08 2020-09-10 Boehringer Ingelheim International Gmbh Anti-il-36r antibody formulations
CN111500668A (zh) * 2020-06-29 2020-08-07 南京诺艾新生物技术有限公司 一种用于测定人IL-36/IL36R/IL1RAcP通路抑制剂的生物学活性的方法
CN112094349A (zh) * 2020-11-04 2020-12-18 上海华奥泰生物药业股份有限公司 靶向于白介素36r的抗体及其制备方法和应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CONNER KIP P., PASTUSKOVAS CINTHIA V., SOTO MARCUS, THOMAS VEENA A., WAGNER MYLO, ROCK DAN A.: "Preclinical characterization of the ADME properties of a surrogate anti-IL-36R monoclonal antibody antagonist in mouse serum and tissues", MABS, LANDES BIOSCIENCE, US, vol. 12, no. 1, 1 January 2020 (2020-01-01), US , XP055955931, ISSN: 1942-0862, DOI: 10.1080/19420862.2020.1746520 *

Also Published As

Publication number Publication date
CN114907477A (zh) 2022-08-16

Similar Documents

Publication Publication Date Title
JP2023075294A (ja) 抗ｃｄ４７抗体及びその応用
CN112625136B (zh) 针对冠状病毒具有中和活性的双特异性抗体及其用途
MX2008014804A (es) Anticuerpos de afinidad elevada a receptor de il-6 humano.
WO2021017071A1 (zh) 抗人st2抗体及其用途
KR20220119394A (ko) Tslp-관련 질환에 대한 치료제의 개발 및 적용
CN113501878B (zh) 针对人tslp的多种抗体及其用途
WO2021139758A1 (zh) 新型多肽复合物
CN111615519B (zh) 结合人il-5的单克隆抗体、其制备方法和用途
CN113651888B (zh) Il-11的抗体及其应用
WO2022174813A1 (zh) 抗GPRC5DxBCMAxCD3三特异性抗体及其用途
US20230035798A1 (en) Antigen Binding Molecules That Bind LIGHT
WO2022166977A1 (zh) 抗人il-36r抗体及其应用
JP7454882B2 (ja) 抗ヒトインターロイキン－４受容体α抗体及びその調製方法並びに使用
WO2021143914A1 (zh) 一种激活型抗ox40抗体、生产方法及应用
CN111662385A (zh) 全人源抗人gpc3单克隆抗体及其用途
CN114395042B (zh) 抗il-33人源化抗体及其应用
JP2021502801A (ja) Ｉｌ−５抗体、その抗原結合フラグメント、およびそれらの医薬適用
CN114426580B (zh) 抗csf-1r抗体及其产品、方法和用途
TWI804099B (zh) 特異性結合糖基化ｃｅａｃａｍ５的抗體及其製備方法
WO2022052968A1 (zh) 针对冠状病毒刺突蛋白的单克隆抗体及其用途
TW202413405A (zh) 抗體、其抗原結合片段及其藥物用途
KR20240022546A (ko) 항il-36r 항체 및 그의 사용
CN117886935A (zh) 抗csf-1r抗体及其应用
WO2023072916A1 (en) Antibodies targeting ccr2
TW202321300A (zh) Il-11人源化抗體及其應用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22749256

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22749256

Country of ref document: EP

Kind code of ref document: A1