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WO2025209572A1 - Anticorps anti-il-1rap, composition pharmaceutique et utilisation associées - Google Patents

Anticorps anti-il-1rap, composition pharmaceutique et utilisation associées

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Publication number
WO2025209572A1
WO2025209572A1 PCT/CN2025/087146 CN2025087146W WO2025209572A1 WO 2025209572 A1 WO2025209572 A1 WO 2025209572A1 CN 2025087146 W CN2025087146 W CN 2025087146W WO 2025209572 A1 WO2025209572 A1 WO 2025209572A1
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WIPO (PCT)
Prior art keywords
seq
antibody
1rap
amino acid
acid sequence
Prior art date
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Pending
Application number
PCT/CN2025/087146
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English (en)
Chinese (zh)
Inventor
王忠民
张鹏
李百勇
夏瑜
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Akeso Pharmaceuticals Inc
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Akeso Biopharma Inc
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Publication of WO2025209572A1 publication Critical patent/WO2025209572A1/fr
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    • 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
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • 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
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention belongs to the field of biomedicine. Specifically, the present invention relates to an anti-IL-1RAP antibody, a pharmaceutical composition thereof, and uses thereof.
  • Acute inflammation is a response to tissue damage or infection caused by dendritic cells or macrophages that secrete pro-inflammatory mediators (such as cytokines). Under normal circumstances, the pathogenic factors are cleared, the inflammatory process ends, the inflammatory cascade is terminated, and the internal environment returns to stability.
  • stimuli such as genetic mutations in senescent cells, bacterial or viral infections, or inflammatory diseases lead to the emergence of chronic inflammation associated with cancer progression.
  • This cancer-related inflammation is divided into two pathways: an intrinsic pathway in which genetics cause inflammation and tumor transformation, and an extrinsic pathway in which inflammation caused by infection or environmental exposure promotes cancer. Both pathways lead to chronic inflammation and play an important role in the initiation, promotion, and proliferation of cancer cells.
  • many cytokines and their pathways are widely overexpressed in different types of cancer and have become potential targets for tumor therapy.
  • the IL-1 superfamily consists of a variety of cytokines and their receptors. IL-1 family members are generally divided into four subfamilies based on their coreceptors: IL-1 (IL-1 ⁇ , IL-1 ⁇ ), IL-33, IL-36 (IL-36 ⁇ , IL-36 ⁇ , and IL-36 ⁇ ), and the IL-18 subfamily, which binds to a different coreceptor (IL-18RAP).
  • IL-1 family members are key signaling molecules in both the innate and adaptive immune systems, mediating a variety of inflammatory responses (Fields James K, Günther Sebastian, Sundberg Eric J, Structural Basis of IL-1 Family Cytokine Signaling. [J]. Front Immunol, 2019, 10:1412).
  • the main receptor and the intracellular TIR domain of IL-1RAP are juxtaposed to recruit and bind to intracellular proteins such as Tollip, MyD88, IRAK family members, TRAF-6, etc., triggering intracellular signaling cascades and inducing the expression of proinflammatory cytokines and chemokines that depend on NF- ⁇ B and AP-1, and participating in various inflammatory and immune responses and cell death.
  • IL-1RAP has been found to be overexpressed in a variety of solid tumors and hematological tumors. Studies have shown that downregulating IL-1RAP in human primary AML cells increased apoptosis and differentiation of AML cells; after introducing the MLL-AF9 fusion oncogene into the hematopoietic stem and progenitor cells of the bone marrow of gene knockout (IL1RAP-/-) mice, it was found that compared with the wild-type (WT) group, the IL1RAP-/- group showed delayed leukemia progression and better survival rate; in the pancreatic cancer (PDAC) cell line A6L, inhibition of IL-1RAP reduced the survival rate and clonogenic ability of PDAC cells.
  • WT wild-type
  • PDAC pancreatic cancer
  • the anti-IL-1RAP antibody or antigen-binding fragment thereof wherein,
  • the variant is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to the corresponding sequence.
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 13
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO: 29;
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:29;
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 29;
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 19, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 29;
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:21, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:29;
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:27, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:29.
  • the anti-IL-1RAP antibody or antigen-binding fragment thereof is selected from Fab, Fab', F(ab') 2 , Fd, Fv, dAb, complementarity determining region fragment, single-chain antibody, humanized antibody or chimeric antibody.
  • the anti-IL-1RAP antibody or antigen-binding fragment thereof wherein,
  • the antibodies include non-CDR regions, and the non-CDR regions are from a species other than murine, such as a human antibody.
  • the anti-IL-1RAP antibody or antigen-binding fragment thereof wherein,
  • the antibody whose constant region is derived from a human antibody
  • the constant region of the antibody is selected from the constant region of human IgG1, IgG2, IgG3, or IgG4.
  • the heavy chain constant region adopts the Ig gamma-1 chain C region, ACCESSION: P01857; the light chain constant region adopts the Ig kappa chain C region, ACCESSION: P01834.
  • the anti-IL-1RAP antibody or its antigen-binding fragment wherein the heavy chain constant region of the antibody is Ig gamma-1 chain C region (for example, the amino acid sequence is shown in SEQ ID NO: 31 or SEQ ID NO: 41); the light chain constant region is Ig kappa chain C region (for example, the amino acid sequence is shown in SEQ ID NO: 35).
  • the anti-IL-1RAP antibody or antigen-binding fragment thereof wherein,
  • the antibody is of human IgG1 subtype
  • the heavy chain constant region of the antibody has the following mutations at position 234, position 235 and/or position 237:
  • amino acid sequence of the heavy chain constant region of the antibody is as shown in SEQ ID NO:33, and the amino acid sequence of the light chain constant region is as shown in SEQ ID NO:35.
  • the anti-IL-1RAP antibody or antigen-binding fragment thereof wherein the EC50 of the anti-IL-1RAP antibody binding to cells expressing IL-1RAP is less than or equal to 10 ⁇ g/mL, less than or equal to 8 ⁇ g/mL, less than or equal to 6 ⁇ g/mL, less than or equal to 4 ⁇ g/mL, or less than or equal to 3 ⁇ g/mL; preferably, the EC50 is measured by FACS (flow cytometry).
  • the anti-IL-1RAP antibody is an anti-IL-1RAP monoclonal antibody.
  • Another aspect of the present invention relates to an isolated or synthetic polypeptide selected from the group consisting of:
  • polypeptide comprising the sequences shown in SEQ ID NOs: 5, 6, and 7, wherein the polypeptide specifically binds to IL-1RAP as part of an anti-IL-1RAP antibody, the antibody further comprising the sequences shown in SEQ ID NOs: 8, 9, and 10;
  • polypeptide comprising the sequences shown in SEQ ID NOs: 8, 9, and 10, wherein the polypeptide specifically binds to IL-1RAP as part of an anti-IL-1RAP antibody, the antibody further comprising the sequences shown in SEQ ID NOs: 5, 6, and 7;
  • polypeptide comprising the sequence shown in SEQ ID NO: 3 or 29, wherein the polypeptide specifically binds to IL-1RAP as part of an anti-IL-1RAP antibody, and the antibody further comprises the sequence shown in SEQ ID NO: 1, 11, 13, 15, 17, 19, 21, 23, 25 or 27.
  • the present invention relates to a biomaterial selected from the group consisting of:
  • nucleic acid molecule encoding the anti-IL-1RAP antibody or antigen-binding fragment thereof, or the isolated or synthesized polypeptide
  • a host cell comprising the nucleic acid molecule or the recombinant vector.
  • Another aspect of the present invention relates to an antibody derivative, which comprises the anti-IL-1RAP antibody or an antigen-binding fragment thereof and is selected from the group consisting of:
  • an antibody conjugate further comprising a coupling portion, preferably the anti-IL-1RAP antibody or antigen-binding fragment thereof is connected to the coupling portion via a linker (for example, the linker is a hydrazone bond, a disulfide bond, or a peptide bond), preferably the coupling portion is a purification tag (such as a His tag), a cytotoxic agent (such as a drug, preferably a compound drug), a detectable label (such as a radioisotope, a luminescent substance, a colored substance, an enzyme), or polyethylene glycol, preferably, the molar ratio of the anti-IL-1RAP antibody or antigen-binding fragment thereof to the compound drug is 1:(2-4), for example, 1:2, 1:3, or 1:4, and
  • Another aspect of the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of the anti-IL-1RAP antibody or antigen-binding fragment thereof, or the antibody derivative according to claim 11.
  • the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.
  • Another aspect of the present invention relates to the use of the anti-IL-1RAP antibody or its antigen-binding fragment, the antibody derivative or the pharmaceutical composition for treating tumors, adverse neurological reactions, autoimmune diseases or inflammatory diseases mediated by IL-1RAP or in the preparation of a medicament for treating tumors, adverse neurological reactions, autoimmune diseases or inflammatory diseases mediated by IL-1RAP.
  • Another aspect of the present invention relates to a method for treating or preventing tumors, comprising administering to a subject in need thereof an effective amount of the anti-IL-1RAP antibody or its antigen-binding fragment, the antibody derivative or the pharmaceutical composition, preferably, the tumor is an IL-1RAP-positive tumor, preferably, the administration is before or after surgery, and/or before or after radiotherapy.
  • the tumor is an IL-1RAP-positive tumor
  • the IL-1RAP-positive tumor is a solid tumor or a blood tumor
  • the solid tumor is: prostate cancer, breast cancer (triple-negative breast cancer), lung cancer (non-small cell lung cancer), colorectal cancer, melanoma, bladder cancer, brain/CNS cancer, bile duct cancer, gallbladder cancer, urothelial cancer, cervical cancer, esophageal cancer, gastric cancer, head/neck cancer, kidney cancer, liver cancer, lymphoma, ovarian cancer, pancreatic cancer, sarcoma, glioma
  • the blood tumor is: multiple myeloma, myeloproliferative disorder (MPD), myelodysplastic syndrome (MDS), leukemia (chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML)).
  • MPD myeloproliferative disorder
  • MDS myel
  • the present invention relates to a method for treating or preventing an autoimmune disease or an inflammatory disease, comprising administering to a subject in need thereof an effective amount of the anti-IL-1RAP antibody or antigen-binding fragment thereof, the antibody derivative, or the pharmaceutical composition of the present invention.
  • the autoimmune disease or inflammatory disease is an IL-1RAP-mediated condition.
  • the administration is before or after surgery, and/or before or after radiotherapy.
  • the autoimmune disease or inflammatory disease is arthritis, ankylosing spondylitis, psoriasis, asthma, atopic dermatitis, systemic lupus erythematosus, chronic obstructive pulmonary disease, inflammatory bowel disease, multiple sclerosis, or irritable bowel syndrome.
  • the present invention relates to a method for treating or preventing adverse neurological reactions, comprising administering to a subject in need thereof an effective amount of the anti-IL-1RAP antibody or antigen-binding fragment thereof, the antibody derivative or the pharmaceutical composition of the present invention.
  • the adverse neurological reaction is a central nervous system adverse reaction or a peripheral nervous system adverse reaction.
  • the peripheral nervous system adverse reaction is chemotherapy-induced peripheral neuropathy.
  • EC50 refers to concentration for 50% of maximal effect, which refers to the concentration that can cause 50% of the maximal effect.
  • the term “antibody” refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having one "light” (L) chain and one "heavy” (H) chain.
  • Antibody light chains can be classified as kappa and lambda light chains.
  • Heavy chains can be classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids.
  • Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of three domains (CH1, CH2, and CH3).
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of one domain, CL.
  • the constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.
  • the VH and VL regions can also be further subdivided into regions of high variability, called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the variable regions (VH and VL) of each heavy chain/light chain pair form the antibody binding site.
  • the assignment of amino acids to regions or domains follows Dondelinger Mathieu , Filée Patrice , Sauvage Eric et al.
  • antibody is not limited to any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies.
  • the antibody can be of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.
  • humanized antibody refers to an antibody or antibody fragment obtained by replacing all or part of the CDR region of a human immunoglobulin (recipient antibody) with the CDR region of a non-human antibody (donor antibody), wherein the donor antibody can be a non-human (e.g., mouse, rat, or rabbit) antibody with the desired specificity, affinity, or reactivity.
  • donor antibody can be a non-human (e.g., mouse, rat, or rabbit) antibody with the desired specificity, affinity, or reactivity.
  • some amino acid residues in the framework region (FR) of the recipient antibody can also be replaced with amino acid residues of the corresponding non-human antibody, or with amino acid residues of other antibodies, to further improve or optimize the performance of the antibody.
  • single chain antibody refers to a molecule comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) connected by a linker.
  • VH antibody heavy chain variable region
  • VL antibody light chain variable region
  • the VL and VH domains are paired to form a monovalent molecule via a linker that enables them to be produced as a single polypeptide chain (see, e.g., Bird et al, Science 1988; 242:423-426 and Huston et al, Proc. Natl. Acad. Sci. USA 1988; 85:5879-5883).
  • the term "host cell” refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, GS cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • the anti-IL-1RAP antibody of the present invention has excellent affinity and specificity for IL-1RAP.
  • the anti-IL-1RAP antibody of the present invention can effectively block the binding of IL-36 ⁇ / ⁇ / ⁇ to IL-36R.
  • the anti-IL-1RAP antibody of the present invention can significantly inhibit the secretion of IL-6 by A549 cells stimulated by IL-1 ⁇ .
  • the anti-IL-1RAP antibody of the present invention can significantly inhibit the secretion of IL-8 by A549-ST2-Racp cells stimulated by IL-33.
  • the anti-IL-1RAP antibody of the present invention can significantly inhibit the secretion of IL-8 by HaCaT cells stimulated by IL-36 ⁇ / ⁇ / ⁇ , among which the inhibition of IL-3 ⁇ activity is more superior.
  • the anti-IL-1RAP antibody of the present invention can inhibit peripheral neuropathy induced by chemotherapy drugs.
  • Figure 4 Affinity test results of 18H6H23L3 (hG1TM) and human IL-1RAP-His, where His contains 6 his.
  • Figure 7 Affinity test results of 18H6H26L3 (hG1TM) and human IL-1RAP-His, where His contains 6 his.
  • Figure 8 Affinity test results of 18H6H27L3 (hG1TM) and human IL-1RAP-His, where His contains 6 his.
  • Figure 9 Affinity test results of 18H6H28L3 (hG1TM) and human IL-1RAP-His, where His contains 6 his.
  • FIG10 Affinity test results of CAN04 and human IL-1RAP-His, wherein the His contains 6 his.
  • FIG23 shows the detection results of the anti-IL-1RAP antibody 18H6 inhibiting IL-6 secretion in the IL-1 ⁇ system.
  • FIG24 shows the results of the binding activity test between the anti-IL-1RAP antibody 18H6 and the antigen IL-1RAP-his.
  • FIG25 Effects of anti-IL-1RAP antibody on paw withdrawal threshold in mice with paclitaxel-induced peripheral neuropathic pain.
  • FIG26 shows the effect of anti-IL-1RAP antibody on body weight in mice with paclitaxel-induced peripheral neuropathy.
  • the 293T-IL-1RAP cell line was constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd.
  • the 293T-IL-1RAP cell line was generated by 293T cells infected with a virus.
  • the virus was prepared using a 3rd Generation Lentiviral System, see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L. J Virol. 1998. 72(11):8463-8471.
  • 293T-NF ⁇ B-Luc (cell line was constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd.
  • the 293T-NF ⁇ B-Luc cell line was prepared by 293T cells infected with viruses.
  • the virus preparation used was 3rd Generation Lentivirus Systems, see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L. J Virol. 1998.
  • lentiviral expression vector used was pCDH-NF ⁇ B-hygro (wherein the vector pCDH- Hygro was obtained based on pCDH-CMV-MCS-EF1-Puro (purchased from Youbao Bio, product number: VT1480).
  • the NF ⁇ B sequence information was referenced from Siggers T, Chang AB, Teixeira A, Wong D, Williams KJ, Ahmed B, Ragoussis J, Udalova IA, Smale ST, Bulyk ML. Principles of dimer-specific gene regulation revealed by a comprehensive characterization of NF- ⁇ B family DNA binding. Nat Immunol. 2011 Nov 20; 13(1): 95-102).
  • Human IL-1 ⁇ (Akesobio, concentration: 3.72 mg/mL, batch number: 20180930); 293T-NF ⁇ B-Luc-ST2 cell line (ST2 UniProt accession number: Q01638) was prepared by infecting 293T cells with viruses.
  • the viruses were prepared using 3rd Generation Lentivirus Systems, see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L. J Virol. 1998. 72(11): 8463-8471.
  • the lentiviral expression vectors used were pNF-kB-Luc2P-hygro (pCDH-Hygro was modified based on pCDH-CMV-MCS-EF1-Puro (purchased from Ubest Biotech, product number: VT1480)) and pCDH-CMV-IL1RL1-FL (pCDH-CMV-Puro was purchased from Ubest Biotech, product number: VT148).
  • IL33-Nhis bio:IL-33 (Uniprot ID:O95760) protein N-terminus is linked to an amino acid tag his and biotin;
  • the 293T-IL36R-Luc cell line (IL36R UniProt accession number: Q9UBH0) was generated by infecting 293T cells with a 3rd Generation Lentivirus System.
  • the virus was prepared using a 3rd Generation Lentivirus Vector System. See, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ , Nguyen M, Trono D, and Naldini L.J Virol.1998.72(11):8463-8471.
  • the lentiviral expression vectors used were pCDH-hIL36RFL-puro (the vector pCDH-CMV-Puro was purchased from U-Bio, product number: VT148) and pCDH-NF ⁇ B-hygro (the vector pCDH-Hygro was modified based on pCDH-CMV-MCS-EF1-Puro (purchased from U-Bio, product number: VT1480)).
  • IL-36gamma/IL-1F9 (aa 18-169, Protein (R&D, Cat. No.: 6835-IL-010);
  • A549 (culture medium: DMEM + 10% FBS);
  • IL-1RAP-6His Human IL-1RAP-6His (IL-1RAP: Accession: Q9NPH3, in which six His tags are attached), also denoted as IL-1RAP-his;
  • the isotype control antibodies used i.e., hIgG1
  • HEL human hen egg lysosomes
  • the variable region sequences of the antibodies are derived from Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies published by Acierno et al. (Acierno et al. J Mol Biol. 2007; 374(1): 130-46.).
  • the constant region fragments of hIgG1 use Ig gamma-1 chain C region, ACCESSION: P01857.1 as the heavy chain constant region (e.g., as shown in SEQ ID NO: 31), and Ig kappa chain C region, ACCESSION: P01834 as the light chain constant region.
  • hIgG1 was produced in the laboratory of Zhongshan Kangfang Biopharmaceutical Co., Ltd. anti-HEL (hG1DM) is hIgG1 (DM), which has L234A and L235A mutations (according to the EU numbering system) in the heavy chain constant region of hIgG1 (for example, as shown in SEQ ID NO: 31).
  • the immunogen used to prepare anti-IL-1RAP antibodies was IL-1RAP-6his (where IL-1RAP is the amino acid sequence shown in Accession: Q9NPH3, connected to six His tags).
  • Splenocytes from immunized mice were fused with mouse myeloma cells to generate hybridoma cells.
  • Hybridomas were screened using IL-1RAP-6his as the antigen using an indirect ELISA method to obtain hybridoma cells that secrete antibodies that specifically bind to IL-1RAP.
  • Stable hybridoma cell lines were obtained from these screened hybridoma cells by limiting dilution. The monoclonal antibodies secreted by these hybridoma cell lines were designated 18H6.
  • the hybridoma cell line obtained above was cultured in CD medium (Chemical Defined Medium, containing 4% Glutamax (Gibco 35050079) and 1% penicillin-streptomycin (Gibco 15140163)) at 5% CO2 and 37°C. After 7 days, the cell culture supernatant was collected, centrifuged, vacuum filtered through a microporous filter, and purified using a HiTrap protein A HP column to produce the antibody 18H6.
  • CD medium Chemical Defined Medium, containing 4% Glutamax (Gibco 35050079) and 1% penicillin-streptomycin (Gibco 15140163)
  • mRNA was extracted from the hybridoma cell line cultured in Preparation 1 according to the method of the Cultured Cell Bacterial Total RNA Extraction Kit (Tiangen, Catalog No. DP430).
  • the PCR amplification product was directly subjected to TA cloning.
  • TA cloning please refer to the instructions of the pEASY-T1 Cloning Kit (Transgen CT101).
  • the nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2, and the fragment is 360bp long.
  • amino acid sequence it encodes is shown in SEQ ID NO:1, with a length of 120 amino acids.
  • the nucleic acid sequence of the light chain variable region is shown in SEQ ID NO: 4, with a length of 336 bp.
  • amino acid sequence it encodes is shown in SEQ ID NO:3, with a length of 112 amino acids.
  • the six CDRs of antibody 18H6 are defined using the IMGT numbering system as follows:
  • the sequence of the heavy chain HCDR1 is shown in SEQ ID NO: 5, the sequence of HCDR2 is shown in SEQ ID NO: 6, and the sequence of HCDR3 is shown in SEQ ID NO: 7;
  • the sequence of the light chain LCDR1 is shown in SEQ ID NO:8, the sequence of LCDR2 is shown in SEQ ID NO:9, and the sequence of LCDR3 is shown in SEQ ID NO:10.
  • the antibody model was simulated by computer, and then the variable region sequences of antibodies 18H6H8L3, 18H6H14L3, 18H6H21L3, 18H6H23L3, 18H6H24L3, 18H6H25L3, 18H6H26L3, 18H6H27L3, and 18H6H28L3 were obtained based on the model (the heavy chain constant region of the antibody all used Ig gamma-1 chain C region, SEQ ID NO: 31; the light chain constant region was Ig kappa chain C region, SEQ ID NO: 35).
  • the nucleic acid sequence length of the heavy chain variable region is 120 bp, and the length of the amino acid sequence encoded therein is 360 aa; the nucleic acid sequence length of the light chain variable region is 112 bp, and the length of the amino acid sequence encoded therein is 336 aa.
  • HCDR1 The sequence of HCDR1 is shown in SEQ ID NO: 5, the sequence of HCDR2 is shown in SEQ ID NO: 6, and the sequence of HCDR3 is shown in SEQ ID NO: 7;
  • the sequence of LCDR1 is shown in SEQ ID NO:8, the sequence of LCDR2 is shown in SEQ ID NO:9, and the sequence of LCDR3 is shown in SEQ ID NO:10.
  • the heavy chain cDNA and light chain cDNA of 18H6H8L3, 18H6H14L3, 18H6H21L3, 18H6H23L3, 18H6H24L3, 18H6H25L3, 18H6H26L3, 18H6H27L3, and 18H6H28L3 were cloned into pUC57simple (provided by GenScript) vector to obtain pUC57simple-18H6H8, pUC57simple-21L3, and pUC57simple-31L3, respectively.
  • endotoxin-free expression plasmids were prepared and transiently transfected into HEK293 cells for antibody expression. After 7 days of culture, the cell culture fluid was collected and affinity purified using a Protein A column to obtain humanized antibodies.
  • Preparation Example 4 Sequence Design of Humanized Antibodies 18H6H8L3 (hG1TM), 18H6H14L3 (hG1TM), 18H6H21L3 (hG1TM), 18H6H23L3 (hG1TM), 18H6H23L3 (hG1TM), 18H6H24L3 (hG1TM), 18H6H25L3 (hG1TM), 18H6H26L3 (hG1TM), 18H6H27L3 (hG1TM), 18H6H28L3 (hG1TM)
  • the present inventors introduced a leucine to alanine point mutation (L234A) at position 234 of the heavy chain constant region, a leucine to alanine point mutation (L235A) at position 235, and a glycine to alanine point mutation (G237A) at position 237 according to the EU numbering system to obtain the mutant humanized antibody 18H6.
  • L234A leucine to alanine point mutation
  • L235A leucine to alanine point mutation
  • G237A glycine to alanine point mutation
  • the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:33; the amino acid sequence of the light chain constant region is shown in SEQ ID NO:35.
  • Example 1 ELISA method for determination of the affinity between anti-IL-1RAP antibody and antigen human IL-1RAP-mFc Binding activity
  • the antigen human IL-1RAP-mFc (mFc amino acid sequence is shown in SEQ ID NO: 43) was coated on the ELISA plate at 1 ⁇ g/ml and incubated at 4°C for 12 hours. After incubation, the antigen-coated ELISA plate was rinsed once with PBST, and then a 1% BSA solution in PBST was used as the blocking solution for the ELISA plate for 2 hours. After the ELISA plate was blocked, the plate was washed 3 times with PBST. Antibodies diluted in a gradient of PBST solution were added to the wells of the ELISA plate. The antibody dilution gradient is detailed in Table 2.
  • the ELISA plate with the antibody to be tested was incubated at 37°C for 30 minutes. After incubation, the plate was washed 3 times with PBST. After washing, a 1:5000 dilution of HRP-labeled goat anti-human IgG Fc (Jackson, catalog number: 109-035-098) secondary antibody working solution was added and incubated at 37°C for 30 minutes. After incubation, wash the plate three times with PBST, then add TMB (Neogen, 308177) and color for 5 minutes in the dark. Stop the color reaction by adding stop solution. Immediately place the plate in a microplate reader and read the OD value of each well at 450 nm. Data were analyzed using SoftMax Pro 6.2.1 software.
  • the binding activity of 18H6H8L3 (hG1TM), 18H6H14L3 (hG1TM), 18H6H24L3 (hG1TM), 18H6H25L3 (hG1TM), 18H6H26L3 (hG1TM), 18H6H27L3 (hG1TM), and 18H6H28L3 (hG1TM) to human IL-1RAP-mFc is comparable to that of the positive drug CAN04 with the same target.
  • Example 2 The kinetic parameters of the binding between the anti-IL-1RAP humanized antibody and hIL-1RAP-6His were determined using the Fortebio molecular interaction instrument.
  • the sample dilution buffer consisted of PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4. 1 ⁇ g/ml of antibody was immobilized on the AHC sensor for 80 seconds. The sensor was equilibrated in the buffer for 60 seconds. The immobilized antibody bound to hIL-1RAP-6His at a concentration of 3.125-50 nM (twofold dilution) for 150 seconds. hIL-1RAP-6His dissociated in the buffer for 500 seconds. The sample plate was vibrated at a rate of 1000 rpm, the detection temperature was 30°C, and the frequency was 5.0 Hz. The data were analyzed using a 1:1 model fit to obtain affinity constants. The data acquisition software was Fortebio Data Acquisition 12.0, and the data analysis software was Fortebio Data Analysis HT 12.0.
  • the affinity constants of anti-IL-1RAP humanized antibodies 18H6H23L3 (hG1TM), 18H6H24L3 (hG1TM), 18H6H25L3 (hG1TM), 18H6H26L3 (hG1TM), 18H6H27L3 (hG1TM), 18H6H28L3 (hG1TM), and CAN04 (as a control antibody) with hIL-1RAP-6His are shown in Table 5, and the test results are shown in Figures 4-10.
  • K D is the affinity constant
  • 293T-IL-1RAP cells were routinely collected, centrifuged at 170 ⁇ g for 5 minutes, and the supernatant was discarded; appropriate amount of PBS was added to resuspend the cells and washed once; the cells were counted and the viability was determined; the cell concentration was adjusted, and the cell suspension was added to a 96-well conical bottom plate at 3*10 5 cells/sample, and an appropriate amount of 1% PBSA (PBS+1% BSA) was added to each well.
  • 1% PBSA PBS+1% BSA
  • the cells were centrifuged at 350 ⁇ g for 5 minutes and the supernatant was discarded; the antibodies were diluted with 1% PBSA to 500, 125, 31.25, 7.81, 1.95, 0.488, 0.0488, 0.00488, and 0.000488 nM, and the diluted antibodies were added to the corresponding samples at 100 ⁇ L/well and the cell pellet was resuspended and incubated on ice for 40 minutes; 150 ⁇ L was added to each well 1% PBSA, centrifuge at 350xg for 5 minutes, discard the supernatant; repeat washing twice, 200 ⁇ L/time; dilute mouse anti-human IgG Fc-Alexa Fluor@647 (500-fold dilution) with 1% PBSA, add the diluted antibody to the corresponding sample at 100 ⁇ L/well and resuspend the cell pellet, place on ice and incubate in the dark for 30 minutes; add 150 ⁇ L 1% PBSA to each well, centrifuge at 350
  • Example 4 Reporter gene assay to detect the blocking effect of anti-IL-1RAP antibodies on the binding of IL-1 ⁇ / ⁇ to IL-1R
  • 293T-NFkB-Luc cells were collected by routine digestion (culture medium: DMEM + 10% FBS, containing hygro: 0.1 mg/mL), centrifuged at 170xg for 5 min, and the supernatant was discarded. The cells were resuspended in DMEM complete culture medium (containing 10% FBS), and the cell density was adjusted. 40 ⁇ L/well was inoculated into a 96-well black plate (containing about 2*10 4 cells/well); dilute the antibody with DMEM complete medium, add the diluted antibody to the corresponding wells at 20 ⁇ L/well, and incubate in an incubator for 30 minutes.
  • the final antibody concentrations are 10, 3.3, 1.1, 0.37, 0.123, 0.041, 0.0137, 0.00137, and 0.000137 ⁇ g/mL. Blank control, negative control, isotype control, and positive control are also designed. Dilute human IL-1 ⁇ / ⁇ protein with DMEM complete medium (final concentration of 0.1 ng/mL), add the diluted protein to the corresponding wells at 20 ⁇ L/well, and incubate in an incubator for 5 hours. Add 50 ⁇ L of Luciferase Assay System to each well, and detect the fluorescence value using a multi-label microplate reader within 5 minutes.
  • the nucleotide sequence of 18H6H8 is:
  • the nucleotide sequence of 18H6H24 is:
  • the nucleotide sequence of 18H6H26 is:
  • the nucleotide sequence of 18H6H27 is:

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Abstract

L'invention concerne un anticorps anti-IL-1RAP et son utilisation. L'anticorps comprend HCDR1 ayant une séquence d'acides aminés telle que représentée dans SEQ ID NO : 5, HCDR2 ayant une séquence d'acides aminés telle que représentée dans SEQ ID NO : 6, et HCDR3 ayant une séquence d'acides aminés telle que représentée dans SEQ ID NO : 7, et LCDR1 ayant une séquence d'acides aminés telle que représentée dans SEQ ID NO : 8, LCDR2 ayant une séquence d'acides aminés telle que représentée dans SEQ ID NO : 9, et LCDR3 ayant une séquence d'acides aminés telle que représentée dans SEQ ID NO : 10.
PCT/CN2025/087146 2024-04-03 2025-04-03 Anticorps anti-il-1rap, composition pharmaceutique et utilisation associées Pending WO2025209572A1 (fr)

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