Anti-il-1RAP antibody, and pharmaceutical composition and use thereof

Abstract

Provided are an anti-IL-1RAP antibody and a use thereof. The antibody comprises HCDR1 having an amino acid sequence as shown in SEQ ID NO: 5, HCDR2 having an amino acid sequence as shown in SEQ ID NO: 6, and HCDR3 having an amino acid sequence as shown in SEQ ID NO: 7, and LCDR1 having an amino acid sequence as shown in SEQ ID NO: 8, LCDR2 having an amino acid sequence as shown in SEQ ID NO: 9, and LCDR3 having an amino acid sequence as shown in SEQ ID NO: 10.

Description

Anti-IL-1RAP antibodies, pharmaceutical compositions and uses thereof Technical Field

[0001] 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. Background Art

[0002] 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. However, 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. In this context, many cytokines and their pathways are widely overexpressed in different types of cancer and have become potential targets for tumor therapy.

[0003] 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-1α, IL-1β), IL-33, IL-36 (IL-36α, IL-36β, and IL-36γ), 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).

[0004] Interleukin-1 receptor accessory protein (IL-1RAP) is a coreceptor for type I interleukin-1 receptor (IL1R1), IL-33R, and IL-36R, and is essential for the transmission of IL-1, IL-33, and IL-36 signaling pathways. The molecular structure of IL-1RAP consists of three immunoglobulin (Ig) extracellular domains that recognize β-trefoil cytokines and a TIR (toll / interleukin-1 receptor) C-terminal endodomain that activates the NF-κB and MAPK pathways. IL-1 family cytokines bind to their respective primary receptors, inducing the recruitment of the coreceptor IL-1RAP to form a complex. 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-κB and AP-1, and participating in various inflammatory and immune responses and cell death.

[0005] 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 primary human 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 clonogenicity of PDAC cells. It also reduced the invasiveness of PDAC and significantly arrested the G0 / G1 phase of the cell cycle. After IL-1RAP downregulation, the level of MAPK involved in the IL-1RAP pathway in cells was also significantly reduced. These results confirm that IL-1RAP plays an important role in tumor development (Frenay Jame, Bellay Pierre-Simon, Oudot Alexandra et al. IL-1RAP, a Key Therapeutic Target in Cancer. [J]. Int J Mol Sci, 2022, 23: 14918).

[0006] As a new class of antibody drugs, anti-IL-1RAP antibodies hold broad application prospects and can be used in immunotherapy for a variety of diseases, including cancer. Nadunolimab (CAN04), developed by Cantargia AB, is a fully humanized, ADCC-enhanced IgG1 antibody that targets IL-1RAP and blocks both IL-1α and IL-1β. Interim data from a Phase I clinical trial released in October 2018 showed a favorable safety profile and stable disease in 38% of patients. A Phase IIa clinical trial is also underway.

[0007] Therefore, it is of great significance to develop antibody drugs with high affinity to IL-1RAP so that they have better therapeutic effects and lower toxic side effects. Summary of the Invention

[0008] After extensive research and creative work, the present inventors have developed an anti-IL-1RAP antibody. Surprisingly, the present inventors have discovered that the anti-IL-1RAP antibody of the present invention (also referred to as the antibody or the antibody of the present invention) has superior affinity and / or specificity, can specifically bind to tumor cells expressing IL-1RAP, and has promising anti-tumor potential. This invention provides the following:

[0009] One aspect of the present invention relates to an anti-IL-1RAP antibody or an antigen-binding fragment thereof, wherein the anti-IL-1RAP antibody comprises HCDR1 to HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO: 1, and LCDR1 to LCDR3 contained in the light chain variable region as shown in SEQ ID NO: 3, wherein preferably: according to the EU numbering system,

[0010] The amino acid sequence of HCDR1 is shown in SEQ ID NO: 5, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 6, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 7, and

[0011] The amino acid sequence of LCDR1 is shown in SEQ ID NO:8, the amino acid sequence of LCDR2 is KVS, shown in SEQ ID NO:9, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:10.

[0012] In some embodiments of the present invention, the anti-IL-1RAP antibody or antigen-binding fragment thereof, wherein,

[0013] The amino acid sequence of the heavy chain variable region of the anti-IL-1RAP antibody is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 and SEQ ID NO: 27, or a variant thereof; and

[0014] The amino acid sequence of the light chain variable region of the anti-IL-1RAP antibody is selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 29, or a variant thereof,

[0015] 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.

[0016] In some embodiments of the present invention, the anti-IL-1RAP antibody or antigen-binding fragment thereof, wherein,

[0017] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 3;

[0018] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 29;

[0019] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 29;

[0020] The 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;

[0021] The 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;

[0022] The 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;

[0023] The 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;

[0024] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 23, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 29;

[0025] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 25, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 29;

[0026] or

[0027] The 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.

[0028] In some embodiments of the present invention, the anti-IL-1RAP antibody or its antigen-binding fragment, wherein the anti-IL-1RAP antibody or its antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragment, single-chain antibody, humanized antibody or chimeric antibody.

[0029] In some embodiments of the present invention, the anti-IL-1RAP antibody or antigen-binding fragment thereof, wherein,

[0030] The antibodies include non-CDR regions, and the non-CDR regions are from a species other than murine, such as a human antibody.

[0031] In some embodiments of the present invention, the anti-IL-1RAP antibody or antigen-binding fragment thereof, wherein,

[0032] The antibody, whose constant region is derived from a human antibody;

[0033] Preferably, the constant region of the antibody is selected from the constant region of human IgG1, IgG2, IgG3 or IgG4. For example, the heavy chain constant region uses the Ig gamma-1 chain C region, ACCESSION: P01857; the light chain constant region uses the Ig kappa chain C region, ACCESSION: P01834.

[0034] In some embodiments of the present invention, the anti-IL-1RAP antibody or antigen-binding fragment thereof, wherein the heavy chain constant region of the antibody is Ig gamma-1 chain C region (e.g., the amino acid sequence shown in SEQ ID NO: 31 or SEQ ID NO: 41); and the light chain constant region is Ig kappa chain C region (e.g., the amino acid sequence shown in SEQ ID NO: 35).

[0035] In some embodiments of the present invention, the anti-IL-1RAP antibody or antigen-binding fragment thereof, wherein,

[0036] The antibody is of human IgG1 subtype,

[0037] Preferably, according to the EU numbering system, based on Ig gamma-1 chain C region, ACCESSION: P01857 (as shown in SEQ ID NO: 31), the heavy chain constant region of the antibody has the following mutations at positions 234, 235 and / or 237:

[0038] L234A and L235A,

[0039] L234A and G237A,

[0040] L235A and G237A,

[0041] or

[0042] L234A, L235A, and G237A;

[0043] Preferably, according to the EU numbering system, the heavy chain constant region of the antibody further has one or more mutations selected from the following:

[0044] N297A, D265A, D270A, P238D, L328E, E233D, H268D, P271G, A330R, C226S, C229S, E233P, P331S, S267E, L328F, A330L, M252Y, S254T, T256E, N297Q, P238S, P238A, A327Q, A327G, P329A, K322A, T394D, G236R, G236A, L328R, A330S, H268A, E318A, and K320A;

[0045] More preferably, the amino acid sequence of the heavy chain constant region of the antibody is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO: 35.

[0046] In some embodiments of the present invention, the anti-IL-1RAP antibody or antigen-binding fragment thereof, wherein the EC binding site of the anti-IL-1RAP antibody to cells expressing IL-1RAP 50 is less than or equal to 10 μg / mL, less than or equal to 8 μg / mL, less than or equal to 6 μg / mL, less than or equal to 4 μg / mL, or less than or equal to 3 μg / mL; preferably, the EC 50 It was measured by FACS (flow cytometry).

[0047] In some embodiments of the present invention, the anti-IL-1RAP antibody is an anti-IL-1RAP monoclonal antibody.

[0048] Another aspect of the present invention relates to an isolated or synthetic polypeptide selected from the group consisting of:

[0049] (1) An isolated or synthesized 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, and the antibody further comprises the sequences shown in SEQ ID NOs: 8, 9, and 10;

[0050] (2) an isolated or synthesized polypeptide comprising the sequence 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 sequence shown in SEQ ID NOs: 5, 6, and 7;

[0051] (3) an isolated or synthesized polypeptide comprising a sequence as shown in SEQ ID NO: 1, 11, 13, 15, 17, 19, 21, 23, 25 or 27, wherein the polypeptide specifically binds to IL-1RAP as part of an anti-IL-1RAP antibody, the antibody further comprising a sequence as shown in SEQ ID NO: 3 or 29; and

[0052] (4) An isolated or synthesized 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.

[0053] In another aspect, the present invention relates to a biomaterial selected from the group consisting of:

[0054] (1) a nucleic acid molecule encoding the anti-IL-1RAP antibody or antigen-binding fragment thereof, or the isolated or synthesized polypeptide,

[0055] (2) a recombinant vector comprising the nucleic acid molecule, and

[0056] (3) A host cell comprising the nucleic acid molecule or the recombinant vector.

[0057] 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:

[0058] (1) 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

[0059] (2) Fusion proteins or multispecific antibodies (such as bispecific antibodies).

[0060] Another aspect of the present invention relates to 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. Optionally, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0061] 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.

[0062] 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. In a specific embodiment, the tumor is an IL-1RAP-positive tumor, preferably, the IL-1RAP-positive tumor is a solid tumor or a blood tumor, preferably, 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, preferably, 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)).

[0063] In another aspect, 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. Preferably, the autoimmune disease or inflammatory disease is an IL-1RAP-mediated condition. Preferably, the administration is before or after surgery, and / or before or after radiotherapy. More preferably, 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.

[0064] In another aspect, 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. Preferably, the adverse neurological reaction is a central nervous system adverse reaction or a peripheral nervous system adverse reaction. Preferably, the peripheral nervous system adverse reaction is chemotherapy-induced peripheral neuropathy.

[0065] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures for cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0066] Based on known antibody numbering systems, such as the Kabat numbering system, the EU numbering system, the Chothia numbering system, the IMGT numbering system, or the AHo numbering system, one of ordinary skill in the art can determine the sequences of the heavy and light chain CDRs based on given antibody heavy and light chain sequences.

[0067] As used herein, the term EC 50 It refers to the concentration for 50% of maximal effect, which is the concentration that can cause 50% of the maximum effect.

[0068] As used herein, 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 μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, 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). 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. Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface / Residue Definition. [J]. Front Immunol, 2018, 9: 2278., or Chothia & Lesk J. Mol. Biol. 1987; 196: 901-917; Chothia et al. Nature 1989; 342: 878-883, or the IMGT numbering system definition, see Ehrenmann F, Kaas Q, Lefranc MP. IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF [J]. Nucleic acids Research, 2009; 38(suppl_1): definition of D301-D307.

[0069] The term "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.

[0070] As used herein, the term "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. In addition, 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. For more details on humanized antibodies, see, for example, Jones et al., Nature, 1986; 321: 522 525; Reichmann et al., Nature, 1988; 332: 323 329; Presta, Curr. Op. Struct. Biol. 1992; 2: 593-596; and Clark, Immunol. Today 2000; 21: 397 402.

[0071] As used herein, the term "single chain antibody (scFv)" refers to a molecule comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) connected by a linker. The VL and VH domains are paired to form a monovalent molecule by 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). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al, Proc. Natl. Acad. Sci. USA 1993; 90: 6444-6448). Other linkers that can be used in the present invention are described by Alfthan et al, Protein Eng. 1995; 8: 725-731, Choi et al, Eur. J. Immunol. 2001; 31: 94-106, Hu et al, Cancer Res. 1996; 56: 3055-3061, Kipriyanov et al, J. Mol. Biol. 1999; 293: 41-56 and Roovers et al, Cancer Immunology, Immunotherapy, 2001, 50(1): 51-59.

[0072] As used herein, 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.

[0073] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its antigen. In certain embodiments, an antibody that specifically binds to an antigen (or has specificity for an antigen) means that the antibody binds to the antigen with a specificity of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7M, 10 -8 M, 10 -9 M or 10 -10 Affinity of M or less (K D ) binds to the antigen.

[0074] As used herein, the term "K D " refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Generally, antibodies bind with a dissociation equilibrium constant of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or less dissociation equilibrium constant (K D ) binding antigen (eg, IL-1RAP protein). K can be determined using methods known to those skilled in the art. D , for example, using the Fortebio molecular interaction instrument.

[0075] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably. In the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0076] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, and is well known in the art and includes, but is not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0077] As used herein, when referring to the amino acid sequence of the human IL-1RAP protein, it includes the full-length IL-1RAP protein (Accession: Q9NPH3), or the extracellular fragment IL-1RAP ECD or a fragment comprising the IL-1RAP ECD; it also includes a fusion protein of the full-length IL-1RAP protein or a fusion protein of the IL-1RAP ECD, such as a fragment fused with the Fc protein fragment (mFc or hFc) of mouse or human IgG. However, those skilled in the art understand that mutations or variations (including but not limited to substitutions, deletions and / or additions) can occur naturally or be artificially introduced into the amino acid sequence of the IL-1RAP protein without affecting its biological function. Therefore, in the present invention, the term "IL-1RAP protein" should include all such sequences, including natural or artificial variants thereof. Furthermore, when describing a sequence fragment of the IL-1RAP protein, it also includes the corresponding sequence fragments in its natural or artificial variants.

[0078] Advantageous Effects of the Invention

[0079] The present invention achieves one or more of the following effects:

[0080] (1) The anti-IL-1RAP antibody of the present invention has excellent affinity and specificity for IL-1RAP.

[0081] (2) The anti-IL-1RAP antibody of the present invention can effectively block the binding of IL-1α / β to IL-1R.

[0082] (3) The anti-IL-1RAP antibody of the present invention can effectively block the binding of IL-33 to IL-33R.

[0083] (4) The anti-IL-1RAP antibody of the present invention can effectively block the binding of IL-36α / β / γ to IL-36R.

[0084] (5) The anti-IL-1RAP antibody of the present invention can significantly inhibit the secretion of IL-6 by A549 cells stimulated by IL-1β.

[0085] (6) 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.

[0086] (7) The anti-IL-1RAP antibody of the present invention can significantly inhibit the secretion of IL-8 by HaCaT cells stimulated by IL-36α / β / γ, among which the inhibition of IL-3γ activity is more superior.

[0087] (8) The anti-IL-1RAP antibody of the present invention can inhibit peripheral neuropathy induced by chemotherapy drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1. Binding activity test results of 18H6H23L3 (hG1TM), CAN04 and antigen IL-1RAP-mFc.

[0089] Figure 2. Binding activity assay results of 18H6H8L3(hG1TM), 18H6H14L3(hG1TM), 18H6H21L3(hG1TM), 18H6H24L3(hG1TM), and CAN04 to human IL-1RAP-mFc.

[0090] Figure 3. Binding activity assay results of 18H6H25L3(hG1TM), 18H6H26L3(hG1TM), 18H6H27L3(hG1TM), 18H6H28L3(hG1TM), and CAN04 to human IL-1RAP-mFc.

[0091] Figure 4. Affinity test results of 18H6H23L3 (hG1TM) and human IL-1RAP-His, where the His contains 6 his chains.

[0092] Figure 5. Affinity test results of 18H6H24L3 (hG1TM) and human IL-1RAP-His, where the His contains 6 his chains.

[0093] Figure 6. Affinity test results of 18H6H25L3 (hG1TM) and human IL-1RAP-His, where the His contains 6 his chains.

[0094] Figure 7. Affinity test results of 18H6H26L3 (hG1TM) and human IL-1RAP-His, where the His contains 6 his chains.

[0095] Figure 8. Affinity test results of 18H6H27L3 (hG1TM) and human IL-1RAP-His, where the His contains 6 his chains.

[0096] Figure 9. Affinity test results of 18H6H28L3 (hG1TM) and human IL-1RAP-His, where the His contains 6 his chains.

[0097] FIG10 . Affinity test results of CAN04 and human IL-1RAP-His, wherein the His contains 6 his.

[0098] Figure 11. Binding activity assay results of 18H6H23L3(hG1TM), 18H6H28L3(hG1TM) and CAN04 to IL-1RAP on the membrane surface of 293T-IL-1RAP cells.

[0099] Figure 12. Detection results of 18H6H23L3 (hG1TM), 18H6H28L3 (hG1TM), and CAN04 blocking the binding of IL-1α to IL-1R.

[0100] Figure 13. Detection results of 18H6H23L3 (hG1TM) and CAN04 blocking the binding of IL-1β to IL-1R.

[0101] Figure 14. Detection results of 18H6H14L3 (hG1TM), 18H6H23L3 (hG1TM), 18H6H28L3 (hG1TM), and CAN04 blocking the binding of IL-33 to IL-33R.

[0102] Figure 15. Detection results of 18H6H14L3 (hG1TM), 18H6H23L3 (hG1TM), 18H6H28L3 (hG1TM), and CAN04 blocking the binding of IL-36α to IL-33R.

[0103] Figure 16. Detection results of 18H6H14L3 (hG1TM), 18H6H23L3 (hG1TM), 18H6H28L3 (hG1TM), and CAN04 blocking the binding of IL-36β to IL-33R.

[0104] Figure 17. Detection results of 18H6H14L3 (hG1TM), 18H6H23L3 (hG1TM), 18H6H28L3 (hG1TM), and CAN04 blocking the binding of IL-36γ to IL-33R.

[0105] FIG18 shows the detection results of anti-IL-1RAP antibody inhibiting IL-6 secretion in IL-1β system.

[0106] FIG19 shows the detection results of anti-IL-1RAP antibody inhibiting IL-8 secretion in IL-33 system.

[0107] Figure 20. Detection results of 18H6H23L3 (hG1TM) and 18H6H28L3 (hG1TM) inhibiting IL-8 secretion in the IL-36α system.

[0108] Figure 21. Detection results of 18H6H23L3 (hG1TM) and 18H6H28L3 (hG1TM) inhibiting IL-8 secretion in the IL-36β system.

[0109] Figure 22. Detection results of 18H6H23L3 (hG1TM) and 18H6H28L3 (hG1TM) inhibiting IL-8 secretion in the IL-36γ system.

[0110] FIG23 shows the detection results of the anti-IL-1RAP antibody 18H6 inhibiting IL-6 secretion in the IL-1β system.

[0111] FIG24 shows the results of binding activity detection between the anti-IL-1RAP antibody 18H6 and the antigen IL-1RAP-his.

[0112] FIG25 . Effects of anti-IL-1RAP antibody on paw withdrawal threshold in mice with paclitaxel-induced peripheral neuropathic pain.

[0113] FIG26 shows the effect of anti-IL-1RAP antibody on body weight in mice with paclitaxel-induced peripheral neuropathy. DETAILED DESCRIPTION

[0114] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0115] The 293T-IL-1RAP cell line was constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd. The 293T-IL-1RAP cell line was prepared by infecting 293T cells with a virus. The virus was prepared using 3rd Generation Lentiviral 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 LJ Virol. 1998. 72(11): 8463-8471. The lentiviral expression vector used was pCDH-hIL-1RAPFL-puro (the vector pCDH-CMV-Puro was purchased from Youbao Bio, product number: VT148, and the hIL-1RAP sequence was Accession Q9NPH3).

[0116] Mouse Anti-Human IgG Fc-Alexa 647 (Southern Biotech, cat. no. 9040-31);

[0117] human IL-1α (R&D, concentration: 0.01 mg / mL);

[0118] Bright-GloTM Luciferase Assay System (Promega, Catalog No.: E2620); Human TNF-α (Sinobio, Catalog No.: GMP.10602-HNAE-50);

[0119] 293T-NFκB-Luc (cell line was constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd. The 293T-NFκB-Luc cell line was prepared by 293T cells infected with viruses. The virus preparation used was 3rd Generation Lentiviral 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 vector used was pCDH-NFκB-hygro (wherein the vector pCDH-Hygro was modified based on pCDH-CMV-MCS-EF1-Puro (purchased from Youbao Bio, catalog number: VT1480). The NFκB sequence information was referenced to 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-κB family DNA binding. Nat Immunol. 2011Nov 20;13(1):95-102);

[0120] Human IL-1β (Akesobio, concentration: 3.72 mg / mL, batch number: 20180930); 293T-NFκB-Luc-ST2 cell line (ST2 UniProt accession number: Q01638) was prepared by infecting 293T cells with viruses. The viruses were prepared using 3rd Generation Lentiviral 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 LJ Virol. 1998. 72(11): 8463-8471. The lentiviral expression vectors used were pNF-kB-Luc2P-hygro (wherein the vector pCDH-Hygro was modified based on pCDH-CMV-MCS-EF1-Puro (purchased from U-Bio, product number: VT1480)) and pCDH-CMV-IL1RL1-FL (wherein the vector pCDH-CMV-Puro was purchased from U-Bio, product number: VT148).

[0121] IL33-Nhis bio: IL-33 (Uniprot ID: O95760) protein N-terminus is linked to an amino acid tag his and biotin;

[0122] The 293T-IL36R-Luc cell line (IL36R UniProt accession number: Q9UBH0) was prepared by infecting 293T cells with a virus. The virus was prepared using a 3rd Generation Lentiviral 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 LJ Virol. 1998. 72(11): 8463-8471. The lentiviral expression vectors used were pCDH-hIL36RFL-puro (wherein the vector pCDH-CMV-Puro was purchased from Ubest Biotech, product number: VT148) and pCDH-NFκB-hygro (wherein the vector pCDH-Hygro was modified based on pCDH-CMV-MCS-EF1-Puro (purchased from Ubest Biotech, product number: VT1480)).

[0123] Recombinant human IL-36α / IL-1F6 (aa 6-158, Protein Summary (R&D, Cat. No. 6995-IL-010);

[0124] Recombinant human IL-36gamma / IL-1F9 (aa 18-169, Protein (R&D, Cat. No. 6835-IL-010);

[0125] Recombinant human IL-36β / IL-1F8 (aa 5-157), Protein Summary (R&D, Cat. No. 6834-ILB-025);

[0126] A549 (culture medium: DMEM + 10% FBS);

[0127] Human IL-6 ELISA Kit (Dakoway, Cat. No.: 1110602);

[0128] A549-ST2-IL1RACP (IL1RACP, also known as IL-1RAP) cell line was prepared by infecting A549 cells with a virus. The virus was prepared using 3rd Generation Lentiviral 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 LJ Virol. 1998. 72(11): 8463-8471. The lentiviral expression vectors used were pCDH-CMV-IL1RL1-FL (wherein the vector pCDH-CMV-Puro was purchased from Ubest Biotech, product number: VT148) and plenti6.3 / V5Topo-vera_IL1RACP (wherein the vector plenti6.3 / V5 Topo was purchased from Invitrogen, product number: K531520).

[0129] Human IL-8 ELISA Kit (Dakoway, catalog number: DKW12-1080-096);

[0130] HaCaT (Guangzhou Jinio Biotechnology Co., Ltd., catalog number: JNO-02001);

[0131] Human IL-1RAP-6His (IL-1RAP: Accession: Q9NPH3, in which six His tags are attached), also denoted as IL-1RAP-his;

[0132] Fortebio Octet molecular interaction system (Manufacturer: Sartorius Model: Octet red96e);

[0133] AHC (Manufacturer: Sartorius Model: 18-5060).

[0134] In the following examples of the present invention, the positive control antibody CAN04 used was produced by Zhongshan Kangfang Biopharmaceutical Co., Ltd., and its heavy chain variable region sequence is shown in SEQ ID NO: 37, and its constant region sequence is shown in SEQ ID NO: 41; its light chain variable region sequence is shown in SEQ ID NO: 39, and its constant region sequence is shown in SEQ ID NO: 35.

[0135] In the following experimental examples of the present invention, the isotype control antibodies used, i.e., hIgG1, are antibodies targeting human hen egg lysosomes (HEL). The variable region sequences of the antibodies are derived from the article "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), i.e., hIgG1 (DM), has L234A and L235A mutations (according to the EU numbering system) in the heavy chain constant region of hIgG1 (e.g., as shown in SEQ ID NO: 31).

[0136] Preparation Example 1: Preparation of anti-IL-1RAP antibody

[0137] 1. Preparation of Hybridoma Cell Lines

[0138] 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.

[0139] 2. Preparation of Anti-IL-1RAP Antibody 18H6

[0140] 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.

[0141] Preparation Example 2: Sequence Analysis of Anti-IL-1RAP Antibody 18H6

[0142] 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).

[0143] According to Invitrogen III First-Strand Synthesis System for RT-PCR Kit Instructions: Synthesize cDNA and perform PCR amplification.

[0144] The PCR amplification product was directly subjected to TA cloning. For specific operations, refer to the instructions of the pEASY-T1 Cloning Kit (Transgen CT101).

[0145] The TA cloned products were directly sequenced, and the sequencing results were as follows:

[0146] The nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2, and the fragment is 360 bp long.

[0147] The amino acid sequence encoded by the gene is shown in SEQ ID NO: 1, and has a length of 120 amino acids.

[0148] The nucleic acid sequence of the light chain variable region is shown in SEQ ID NO: 4, and is 336 bp in length.

[0149] The amino acid sequence encoded by the gene is shown in SEQ ID NO: 3, and has a length of 112 amino acids.

[0150] The six CDRs of antibody 18H6 are defined using the IMGT numbering system as follows:

[0151] 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;

[0152] 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.

[0153] Preparation Example 3: Design and Preparation of Light and Heavy Chains of Humanized Antibodies Against Human IL-1RAP

[0154] 1. Light and heavy chain design of humanized anti-human IL-1RAP antibodies 18H6H8L3, 18H6H14L3, 18H6H21L3, 18H6H23L3, 18H6H24L3, 18H6H25L3, 18H6H26L3, 18H6H27L3, and 18H6H28L3

[0155] According to the sequence of antibody 18H6 obtained in Preparation Example 1, an 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 used Ig kappa chain C region, SEQ ID NO: 35).

[0156] The designed variable region sequences are shown in Table 1 below.

[0157] Table 1

[0158] The above 9 antibodies 18H6H8L3, 18H6H14L3, 18H6H21L3, 18H6H23L3, 18H6H24L3, 18H6H25L3, 18H6H26L3, 18H6H27L3, and 18H6H28L3, 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.

[0159] And the above 9 antibodies have the same HCDR1-HCDR3 and LCDR1-LCDR3, as follows:

[0160] 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;

[0161] 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.

[0162] 2. Preparation of humanized antibodies 18H6H8L3, 18H6H14L3, 18H6H21L3, 18H6H23L3, 18H6H24L3, 18H6H25L3, 18H6H26L3, 18H6H27L3, and 18H6H28L3

[0163] The heavy chain constant region all used the Ig gamma-1 chain C region, SEQ ID NO: 31; the light chain constant region all used the Ig kappa chain C region, SEQ ID NO: 35.

[0164] 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. -18H6L3; pUC57simple-18H6H14, pUC57simple-18H6H21, pUC57simple-18H6H23, pUC57simple-18H6 H24, pUC57simple-18H6H25, pUC57simple-18H6H26, pUC57simple-18H6H27, pUC57simple-18H6H28.

[0165] Referring to the standard techniques described in the Molecular Cloning Experimental Guide (Second Edition), the full-length heavy and light chain genes synthesized by EcoRI & HindIII digestion were subcloned into the expression vector pcDNA3.1 by restriction enzyme digestion (EcoRI & HindIII) to obtain expression plasmids pcDNA3.1-18H6H8, pcDNA3.1-18H6H14, pcDNA3.1-18H6H21, pcDNA3.1-18H6H23, pcDNA3.1-18H6H24, pcDNA3.1-18H6H25, pcDNA3.1-18H6H26, pcDNA3.1-18H6H27, pcDNA3.1-18H6H28, and the heavy / light chain genes of the recombinant expression plasmids were further sequenced and analyzed. Subsequently, the recombinant plasmids containing the corresponding light and heavy chain gene combinations (pcDNA3.1-18H6H8, pcDNA3.1-18H6H14, pcDNA3.1-18H6H21, pcDNA3.1-18H6H23, pcDNA3.1-18H6H24, pcDNA3.1-18H6H25, pcDNA3.1-18H6H26, pcDNA3.1-18H6H27, pcDNA3.1-18H6H28) were designed. H8 / pcDNA3.1-18H6L3, pcDNA3.1-18H6H14 / pcDNA3.1-18H6L3, pcDNA3.1-18H6H21 / pcDNA3. 1-18H6L3, pcDNA3.1-18H6H23 / pcDNA3.1-18H6L3, pcDNA3.1-18H6H24 / pcDNA3.1-18H6L3, p cDNA3.1-18H6H25 / pcDNA3.1-18H6L3, pcDNA3.1-18H6H26 / pcDNA3.1-18H6L3, pcDNA3.1-18H6H27 / pcDNA3.1-18H6L3, and pcDNA3.1-18H6H28 / pcDNA3.1-18H6L3) were co-transfected into 293F cells, and the culture medium was collected for purification. After sequencing verification, endotoxin-free expression plasmids were prepared and transiently transfected into HEK293 cells for antibody expression. After 7 days of culture, the cell culture medium was collected and affinity purified using a Protein A column to obtain humanized antibodies.

[0166] 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)

[0167] Based on the humanized antibody obtained in Preparation Example 3, the present inventors prepared the humanized antibody according to the EU numbering system. system), by introducing a leucine to alanine point mutation (L234A) at position 234, a leucine to alanine point mutation (L235A) at position 235, and a glycine to alanine point mutation (G237A) at position 237 of the heavy chain constant region, the mutant humanized antibodies 18H6H8L3 (hG1TM), 18H6H14L3 (hG1TM), 18H6H21L3 (hG1TM), 18H6H23L3 (hG1TM), 18H6H23L3 (hG1TM), 18H6H24L3 (hG1TM), 18H6H25L3 (hG1TM), 18H6H26L3 (hG1TM), 18H6H27L3 (hG1TM), and 18H6H28L3 (hG1TM) were obtained. The amino acid sequence of its heavy chain constant region is shown in SEQ ID NO: 33; the amino acid sequence of its light chain constant region is shown in SEQ ID NO: 35.

[0168] Example 1: ELISA method to determine the binding activity of anti-IL-1RAP antibody and antigen human IL-1RAP-mFc

[0169] Experimental Procedure: 1 μg / ml of the antigen human IL-1RAP-mFc (mFc amino acid sequence as shown in SEQ ID NO:43) was coated onto an ELISA plate and incubated at 4°C for 12 hours. Following incubation, the plate was rinsed once with PBST and then blocked with 1% BSA in PBST for 2 hours. After blocking, the plate was washed three times with PBST. Antibodies were added to the wells of the plate in a serial dilution of PBST. The antibody dilution series is detailed in Table 2. The plate with the test antibodies was incubated at 37°C for 30 minutes. After incubation, the plate was washed three times with PBST. After washing, a 1:5000 dilution of HRP-conjugated goat anti-human IgG Fc (Jackson, Cat. No. 109-035-098) secondary antibody was added and incubated at 37°C for 30 minutes. After incubation, the plate was washed three times with PBST. TMB (Neogen, 308177) was then added and color was developed for 5 minutes in the dark. The color reaction was terminated by adding stop solution. The plate was immediately placed in a microplate reader and the OD values ​​of each well were read at 450 nm. Data were analyzed using SoftMax Pro 6.2.1 software.

[0170] The OD values ​​of each dose of anti-IL-1RAP antibody binding to the antigen human IL-1RAP-mFc are shown in Tables 2-4. The antibody concentration is used as the horizontal axis and the absorbance value is used as the vertical axis to fit the curve and calculate the antibody binding EC50 The results are shown in Table 2-4 and Figure 1-3 below.

[0171] The experimental results showed that antibodies 18H6H23L3 (hG1TM), 18H6H8L3 (hG1TM), 18H6H14L3 (hG1TM), 18H6H21L3 (hG1TM), 18H6H24L3 (hG1TM), 18H6H25L3 (hG1TM), 18H6H26L3 (hG1TM), 18H6H27L3 (hG1TM), and 18H6H28L3 (hG1TM) can all effectively bind to human IL-1RAP-mFc, and the binding efficiency is dose-dependent.

[0172] The above experimental results show that under the same experimental conditions, the binding activity of 18H6H23L3 (hG1TM) and 18H6H21L3 (hG1TM) to human IL-1RAP-mFc is better than that of the positive drug CAN04 with the same target. 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.

[0173] Table 2. Binding activity assay results of 18H6H23L3 (hG1TM) and CAN04 with human IL-1RAP-mFc

[0174] Table 3. Binding activity assay results of 18H6H8L3(hG1TM), 18H6H14L3(hG1TM), 18H6H21L3(hG1TM), 18H6H24L3(hG1TM), and CAN04 to human IL-1RAP-mFc

[0175] Table 4. Binding activity assay results of 18H6H25L3(hG1TM), 18H6H26L3(hG1TM), 18H6H27L3(hG1TM), 18H6H28L3(hG1TM), and CAN04 to human IL-1RAP-mFc

[0176] 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.

[0177] The sample dilution buffer consisted of PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4. Antibody was immobilized on the AHC sensor at 1 μg / ml for 80 seconds. The sensor was equilibrated in the buffer for 60 seconds. The sensor-immobilized antibody bound to hIL-1RAP-6His at concentrations ranging from 3.125 to 50 nM (two-fold dilutions) for 150 seconds, and hIL-1RAP-6His dissociated in the buffer for 500 seconds. The sample plate was shaken at a rate of 1000 rpm, the detection temperature was 30°C, and the frequency was 5.0 Hz. Data were analyzed using a 1:1 model fit to obtain affinity constants. Data acquisition software was Fortebio Data Acquisition 12.0, and data analysis software was Fortebio Data Analysis HT 12.0.

[0178] 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.

[0179] The experimental results are shown in Table 5 and Figures 4-10. The affinity constants of humanized antibodies 18H6H23L3 (hG1TM), 18H6H24L3 (hG1TM), 18H6H25L3 (hG1TM), 18H6H26L3 (hG1TM), 18H6H27L3 (hG1TM), 18H6H28L3 (hG1TM), and CAN04 to human hIL1R3-6His are 5.19E-10M, 8.79E-10M, 8.39E-10M, 4.65E-10M, 8.49E-10M, 5.24E-10M, and 6.82E-10M, respectively.

[0180] The above experimental results show that the binding ability of 18H6H23L3(hG1TM), 18H6H24L3(hG1TM), 18H6H25L3(hG1TM), 18H6H26L3(hG1TM), 18H6H27L3(hG1TM), and 18H6H28L3(hG1TM) is comparable to that of the positive control antibody CAN04, indicating that the humanized antibodies 18H6H23L3(hG1TM), 18H6H24L3(hG1TM), 18H6H25L3(hG1TM), 18H6H26L3(hG1TM), 18H6H27L3(hG1TM), and 18H6H28L3(hG1TM) all have strong binding ability to hIL-1RAP-6His.

[0181] Table 5. Affinity constants of anti-IL-1RAP humanized antibodies and hIL-1RAP-6His

[0182] K D is the affinity constant; K D =kdis / kon

[0183] Example 3: FACS detection of the binding ability of anti-IL-1RAP antibodies

[0184] 293T-IL-1RAP cells were routinely collected and centrifuged at 170×g for 5 min, and the supernatant was discarded. Appropriate amount of PBS was added to resuspend the cells and washed once. Cell count and viability were determined. Cell concentration was adjusted and the cell suspension was diluted to 3×10 5 Cells / samples were added to a 96-well conical bottom plate, and an appropriate amount of 1% PBSA (PBS + 1% BSA) was added to each well. The cells were centrifuged at 350 x g for 5 min and the supernatant was discarded. 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. The diluted antibodies were added to the corresponding samples at 100 μL / well and the cell pellet was resuspended. The cells were incubated on ice for 40 min. 150 μL of 1% PBSA was added to each well, and the cells were centrifuged at 350 x g for 5 min. The supernatant was discarded. The wash was repeated twice, 200 μL / times. Mouse anti-human IgG Fc-Alexa Fluor 500 was diluted with 1% PBSA. Fluor@647 (500-fold dilution), add the diluted antibody to the corresponding sample at 100 μL / well and resuspend the cell pellet, incubate on ice in the dark for 30 minutes; add 150 μL 1% PBSA to each well, centrifuge at 350xg for 5 minutes, and discard the supernatant; repeat the wash twice, 200 μL / time; add 200 μL 1% PBSA to each well, resuspend the cell pellet, transfer to a sample tube, and analyze on the instrument.

[0185] The experimental results are shown in Table 6 and Figure 11. Under the same experimental conditions, the binding of 18H6H23L3 (hG1TM), 18H6H28L3 (hG1TM), and CAN04 to IL-1RAP on the surface of 293T-IL-1RAP cell membrane was dose-dependent. 50 The results showed that both 18H6H23L3 (hG1TM) and 18H6H28L3 (hG1TM) could effectively bind to IL-1RAP, and their binding activity was comparable to that of the positive drug CAN04 with the same target.

[0186] Table 6. FACS detection of the ability of 18H6H23L3 (hG1TM), 18H6H28L3 (hG1TM) and CAN04 to bind to IL1R3

[0187] Example 4: Reporter gene assay to detect the blocking effect of anti-IL-1RAP antibodies on the binding of IL-1α / β to IL-1R

[0188] 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 μL / 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 μL / 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 μg / mL. Blank, negative, isotype, and positive controls are also designed. Dilute human IL-1α / β protein with DMEM complete medium (final concentration of 0.1 ng / mL), add the diluted protein to the corresponding wells at 20 μL / well, and incubate in an incubator for 5 hours. Add 50 μL of Luciferase Assay System to each well, and detect fluorescence values ​​using a multi-label microplate reader within 5 minutes.

[0189] The experimental results of anti-IL-1RAP antibodies blocking the binding of IL-1α to IL-1R are shown in Table 7 and Figure 12. Under the same experimental conditions, 18H6H23L3 (hG1TM), 18H6H28L3 (hG1TM), and CAN04 blocked the binding of IL-1α to IL-1R in a dose-dependent manner, with EC50 values ​​of 0.09478 nM, 0.0944 nM, and 0.1034 nM, respectively. Furthermore, the inhibition rates of 18H6H23L3 (hG1TM) and 18H6H28L3 (hG1TM) against the binding of IL-1α to IL-1R were significantly higher than that of the same-target positive drug CAN04. ​​These results indicate that 18H6H23L3 (hG1TM) and 18H6H28L3 (hG1TM) have superior blocking activities against the binding of IL-1α to IL-1R than the same-target positive drug CAN04.

[0190] The experimental results of anti-IL-1RAP antibody blocking IL-1β binding to IL-1R are shown in Table 8 and Figure 13. Under the same experimental conditions, 18H6H23L3 (hG1TM) and CAN04 blocked IL-1β binding to IL-1R in a dose-dependent manner, with EC50 values ​​of 0.04317 nM and 0.1180 nM, respectively. These results demonstrate that 18H6H23L3 (hG1TM) significantly outperforms CAN04, a positively charged drug for the same target, in inhibiting IL-1β binding to IL-1R.

[0191] Table 7. 18H6H23L3 (hG1TM), 18H6H28L3 (hG1TM), and CAN04 block IL-1α binding to IL-1R

[0192] Table 8. 18H6H23L3 and CAN04 block the binding of IL-1β to IL-1R

[0193] Example 5: Reporter gene assay to detect the blocking effect of anti-IL-1RAP antibodies on the binding of IL-33 to IL-33R

[0194] 293T-NFkB-Luc-ST2 cells were collected by routine digestion (culture medium: DMEM + 10% FBS, containing hygro: 0.1 mg / mL, Puro: 2 μg / mL), centrifuged at 170×g 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 μL / well was inoculated into a 96-well black plate (containing about 1*10 4cells / well); dilute the antibody with DMEM complete medium, add the diluted antibody to the corresponding wells at 20 μL / well, and incubate in an incubator for 30 minutes. The final antibody concentrations were 30, 10, 3.3, 1.1, 0.37, 0.123, 0.041, 0.0137, and 0.00137 μg / mL. Blank, negative, isotype, and positive controls were also designed. Dilute human IL-33 protein with DMEM complete medium (final concentration of 0.002 ng / mL) and add the diluted protein to the corresponding wells at 20 μL / well. Incubate in an incubator for 5 hours. Add 50 μL of Luciferase Assay System to each well, and detect fluorescence values ​​using a multi-label microplate reader within 5 minutes.

[0195] The experimental results are shown in Table 9 and Figure 14. Under the same experimental conditions, 18H6H23L3 (hG1TM), 18H6H28L3 (hG1TM), 18H6H14L3 (hG1TM) and CAN04 blocked the binding of IL-33 to IL-33R in a dose-dependent manner. 50 The inhibitory rates of 18H6H14L3 (hG1TM), 18H6H23L3 (hG1TM), and 18H6H28L3 (hG1TM) against IL-33 binding to IL-33R were significantly higher than those of the same-target drug, CAN04. ​​The results showed that the blocking activities of 18H6H23L3 (hG1TM) and 18H6H28L3 (hG1TM) for IL-33 binding to IL-33R were superior to those of the same-target drug, CAN04, while the blocking activity of 18H6H14L3 for IL-33 binding to IL-33R was comparable to that of the same-target drug, CAN04.

[0196] Table 9. 18H6H14L3 (hG1TM), 18H6H23L3 (hG1TM), 18H6H28L3 (hG1TM), and CAN04 block IL-33 binding to IL-33R

[0197] Example 6: Reporter gene assay to detect the blocking effect of anti-IL-1RAP antibodies on the binding of IL-36α / β / γ to IL-36R

[0198] 293T-IL36R-Luc cells were collected by routine digestion (culture medium: DMEM + 10% FBS, containing hygro: 0.1 mg / mL, Puro: 2 μg / mL), centrifuged at 170×g 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 to 200,000 / mL. 40 μL / well was inoculated into a 96-well black plate (containing about 1*104 cells / well); dilute the antibody with DMEM complete medium, add the diluted antibody to the corresponding wells at 20 μL / well, and incubate in an incubator for 30 minutes. The final antibody concentrations are 300, 100, 33.3, 11.1, 3.7, 1.23, 0.41, 0.041, 0.0041, and 0.00041 nM. Blank, negative, isotype, and positive controls are also designed. Dilute human IL-36α / β / γ protein with DMEM complete medium, add the diluted protein to the corresponding wells at 20 μL / well, and the working concentration of IL-36α / β / γ protein is 1 ng / mL. Incubate in an incubator for 5 hours; add 50 μL of Luciferase Assay System to each well, and detect fluorescence values ​​using a multi-label microplate reader within 5 minutes.

[0199] The experimental results are shown in Tables 10-11 and Figures 15-17. Under the same experimental conditions, 18H6H14L3 (hG1TM), 18H6H23L3 (hG1TM), 18H6H28L3 (hG1TM) and CAN04 blocked the binding of IL-36α to IL-36R in a concentration-dependent manner. 50 The inhibitory rates of 18H6H14L3 (hG1TM), 18H6H23L3 (hG1TM), and 18H6H28L3 (hG1TM) in inhibiting the binding of IL-36α to IL-36R were significantly higher than those of the positive drug CAN04 with the same target. The results showed that the ability of 18H6H14L3 (hG1TM), 18H6H23L3 (hG1TM), and 18H6H28L3 (hG1TM) to block the binding of IL-36α to IL-36R was significantly better than that of the positive drug CAN04 with the same target.

[0200] Under the same experimental conditions, 18H6H14L3 (hG1TM), 18H6H23L3 (hG1TM), 18H6H28L3 (hG1TM) and CAN04 blocked the binding of IL-36β to IL-36R in a concentration-dependent manner. 50The inhibitory rates of 18H6H14L3 (hG1TM), 18H6H23L3 (hG1TM), and 18H6H28L3 (hG1TM) in inhibiting the binding of IL-36β to IL-36R were significantly higher than those of the positive drug CAN04 with the same target. The results showed that the ability of 18H6H14L3 (hG1TM), 18H6H23L3 (hG1TM), and 18H6H28L3 (hG1TM) to block the binding of IL-36β to IL-36R was significantly better than that of the positive drug CAN04 with the same target.

[0201] Under the same experimental conditions, 18H6H14L3 (hG1TM), 18H6H23L3 (hG1TM), 18H6H28L3 (hG1TM) and CAN04 blocked the binding of IL-36γ to IL-36R in a concentration-dependent manner. 50 The inhibitory rates of 18H6H14L3 (hG1TM), 18H6H23L3 (hG1TM), and 18H6H28L3 (hG1TM) in inhibiting the binding of IL-36γ to IL-36R were significantly higher than those of the positive drug CAN04 with the same target. The results showed that the ability of 18H6H14L3 (hG1TM), 18H6H23L3 (hG1TM), and 18H6H28L3 (hG1TM) to block the binding of IL-36γ to IL-36R was significantly better than that of the positive drug CAN04 with the same target.

[0202] Table 10. 18H6H14L3 (hG1TM), 18H6H23L3 (hG1TM), 18H6H28L3 (hG1TM), and CAN04 block IL-36α / β / γ binding to IL-36R

[0203] Table 11. Inhibition rate of 18H6H14L3, 18H6H23L3, 18H6H28L3 and CAN04 in blocking the binding of IL-36α / β / γ to IL-36R

[0204] Example 7: Anti-IL-1RAP Antibody Inhibits Cytokine Secretion

[0205] 1. Anti-IL-1RAP antibodies inhibit IL-6 secretion in the IL-1β system

[0206] A549 cells were seeded into 96-well plates at 6*10 3Cells were cultured for 24 hours and dosed when the cell confluence reached 80%. Antibodies were added (at a final concentration gradient of 0.065, 0.52, 4.16, 33.3, 100, 300, and 900 nM, or at a final concentration gradient of 0.3, 30, and 300 nM) to the cells and incubated at 37°C for 30 minutes. IL-1β (final concentration 0.01 ng / mL) was then added for 24 hours. The supernatant was collected and assayed with an IL-6 ELISA kit.

[0207] The results are shown in Table 12 and Figure 18. Under the same experimental conditions, 18H6H23L3 (hG1TM), 18H6H26L3 (hG1TM), 18H6H27L3 (hG1TM) and 18H6H28L3 (hG1TM) could significantly inhibit the secretion of IL-6 from A549 cells stimulated by IL-1β. The inhibitory activity was concentration-dependent. EC 50 The results showed that 18H6H23L3(hG1TM), 18H6H26L3(hG1TM), 18H6H27L3(hG1TM) and 18H6H28L3(hG1TM) had the same inhibitory effect on IL-6 secretion from A549 cells as the positive drug CAN04 for the same target.

[0208] The results are shown in Figure 23. Under the same experimental conditions, mouse antibodies 18H6 and CAN04 significantly inhibited IL-1β-stimulated IL-6 secretion from A549 cells. The inhibitory activity was concentration-dependent, and antibody 18H6 was more potent than CAN04, a positive drug for the same target, in inhibiting IL-1β-stimulated IL-6 secretion from A549 cells.

[0209] Table 12. Anti-IL-1RAP antibodies inhibit IL-6 secretion in the IL-1β system

[0210] 2. Anti-IL-1RAP antibodies inhibit IL-8 secretion in the IL-33 system

[0211] A549-ST2-Racp (i.e., A549-ST2-IL1RACP) cells were seeded into 96-well plates at 6*10 3 Cells were cultured for 24 hours and dosed when cell confluence reached 80%. Antibodies (final concentrations of 1800, 360, 72, 14.4, 2.62, and 0.262 nM, respectively) were added to the cells and incubated at 37°C for 30 minutes. IL-33 (final concentration 0.1 nM) was then added for 24 hours. The supernatant was collected and assayed with an IL-8 ELISA kit.

[0212] The results are shown in Table 13 and Figure 19. Under the same experimental conditions, 18H6H8L3 (hG1TM), 18H6H23L3 (hG1TM), 18H6H26L3 (hG1TM) and 18H6H28L3 (hG1TM) could significantly inhibit the secretion of IL-8 by A549-ST2-Racp cells stimulated by IL-33. The inhibitory activity was concentration-dependent. EC 50 5.134×10 -13 The results showed that 18H6H8L3(hG1TM), 18H6H23L3(hG1TM), 18H6H26L3(hG1TM) and 18H6H28L3(hG1TM) were significantly better than CAN04, a positive drug for the same target, in inhibiting IL-8 secretion from A549-ST2-Racp cells stimulated by IL-33.

[0213] Table 13. Anti-IL-1RAP antibodies inhibit IL-8 secretion in the IL-33 system

[0214] 3. Anti-IL-1RAP antibodies inhibit IL-8 secretion in the IL-36 system

[0215] HaCaT (DMEM + 10% FBS) cells were digested and plated (3.5*10 4 Cells were plated and plated with 100 μL per well for 3 hours. For isotype control and blank groups, 50 μL of diluted antibodies (0.16, 0.8, 4, 20, and 100 nM) were added to the corresponding wells and incubated in a cell culture incubator for 30 minutes. IL36α / IL36β / IL36γ (0.5 nM) (all purchased from R&D) were added to the corresponding wells and incubated overnight in a cell culture incubator. The cell plate was centrifuged, and the supernatant was collected for preliminary testing and final analysis using the IL8 kit.

[0216] The experimental results are shown in Figures 20-22. Under the same experimental conditions, 18H6H23L3 (hG1TM), 18H6H28L3 (hG1TM), and CAN04 can all significantly inhibit IL-8 secretion from HaCaT cells stimulated by IL-36α\β in a concentration-dependent manner, and the inhibitory activity is comparable to that of CAN04.

[0217] Under the same experimental conditions, 18H6H23L3 (hG1TM), 18H6H28L3 (hG1TM) and CAN04 could significantly inhibit the secretion of IL-8 by HaCaT cells stimulated by IL-36γ in a concentration-dependent manner, and the inhibitory activity was significantly better than that of CAN04.

[0218] Example 8: ELISA method to determine the binding activity of anti-IL-1RAP antibody and antigen human IL-1RAP-his

[0219] Experimental Procedure: ELISA plates were coated with 1 μg / ml of the antigen human IL-1RAP-his and incubated at 4°C for 12 hours. Following incubation, the coated plates were rinsed once with PBST and then blocked with 1% BSA in PBST for 2 hours. After blocking, the plates were washed three times with PBST. Antibodies were serially diluted in PBST and added to the wells. The antibody dilution series is shown in Table 2. The plates were incubated at 37°C for 30 minutes and washed three times with PBST. After washing, HRP-conjugated goat anti-mouse IgG Fc (Jackson, Cat. No. 105-035-071) and HRP-conjugated goat anti-human IgG Fc (Jackson, Cat. No. 109-035-098) secondary antibodies were added at a 1:5000 dilution and incubated at 37°C for 30 minutes. After incubation, the plate was washed four times with PBST, then TMB (Neogen, 308177) was added and color was developed for 4 minutes in the dark. The color reaction was terminated by adding stop solution. The plate was immediately placed in a microplate reader and the OD values ​​of each well were read at 450 nm. Data were analyzed using SoftMax Pro 6.2.1 software.

[0220] The OD values ​​of each dose of anti-IL-1RAP antibody binding to the antigen human IL-1RAP-his are shown in Table 14. The antibody concentration was used as the abscissa and the absorbance value as the ordinate to fit the curve and calculate the antibody binding EC 50 , the results are shown in Table 14 and Figure 24 below.

[0221] The experimental results showed that antibody 18H6 can effectively bind to human IL-1RAP-his, and the binding efficiency is dose-dependent.

[0222] The above experimental results show that under the same experimental conditions, the binding activity of antibody 18H6 to human IL-1RAP-his is equivalent to that of the positive drug CAN04 with the same target.

[0223] Table 14. Binding activity test results of mouse antibodies 18H6 and CAN04 to human IL-1RAP-his

[0224] Example 9: In vivo pharmacodynamic activity detection of anti-IL-1RAP antibodies

[0225] To investigate the analgesic efficacy of anti-IL-1RAP antibodies in a chemotherapy-induced peripheral neuropathy (CIPN) model, 6-week-old female B-hIL1RAcP transgenic mice (Biocytogen Incorporated, Jiangsu Gene Biotechnology Co., Ltd.) were treated with paclitaxel to establish a CIPN model. The paw withdrawal threshold (PWT) of B-hIL1RAcP transgenic mice was measured after treatment with anti-IL-1RAP antibodies. On day 12 after injection of paclitaxel (Bristol-Myers Squibb, lot number 3B07258, 6 mg / mL), mice were randomly divided into four groups (n=8) based on their PWT values ​​(measured using a Von-Frey fiber pain threshold test kit, North Coast Medical, USA) to form a control group, a high-dose anti-IL-1RAP antibody group, a medium-dose anti-IL-1RAP antibody group, and a low-dose anti-IL-1RAP antibody group. The day of group assignment was defined as day 0 (D0). On the first day (D1) after the model mice were grouped, the isotype control group was intraperitoneally injected with isotype control anti-HEL (hG1DM), and the high-, medium-, and low-dose anti-IL-1RAP antibody groups were intraperitoneally injected with corresponding doses of 18H6H23L3 (hG1TM), twice a week, for a total of 5 times (i.e., D1, D4, D7, D10, and D15); the experimental scheme is detailed in Table 15.

[0226] Table 15: Dosage regimen of anti-IL-1RAP antibody analgesic efficacy in chemotherapy-induced peripheral neuropathic pain model

[0227] The results are shown in Figures 25-26. The results showed that after group administration of 18H6H23L3 (hG1TM) to CIPN model mice, PWT increased in all 18H6H23L3 (hG1TM) dose groups compared to the anti-HEL (hG1DM) isotype control group. 18H6H23L3 (hG1TM) 60 mg / kg was the most effective, with PWT values ​​significantly different from the isotype control group at 2 days after the first dose (D3), 2 days after the third dose (D9), 4 days after the fourth dose (D14), and 2 days after the fifth dose (D17) (P < 0.05, P < 0.001, P < 0.01, and P < 0.01). The PWT value of 18H6H23L3 (hG1TM) 30 mg / kg was significantly different from the isotype control group at 2 days after the third dose (D9) (P < 0.01), and the PWT values ​​of the dose groups were dose-dependent.

[0228] In addition, CIPN model mice tolerated the test drugs well, and there was no significant weight loss in all groups of mice.

[0229] Sequence Listing (wherein the underline represents the CDR sequence):

[0230] Amino acid sequence of 18H6-VH:

[0231] Nucleotide sequence of 18H6-VH:

[0232] Amino acid sequence of 18H6-VL:

[0233] Nucleotide sequence of 18H6-VL:

[0234] CDR sequence of 18H6:

[0235] HCDR1: GFSFTDYI (SEQ ID NO: 5)

[0236] HCDR2: INPNYGST (SEQ ID NO: 6)

[0237] HCDR3:AIFYGWDYYAMDY(SEQ ID NO:7)

[0238] LCDR1:QSIVQSNGYAY(SEQ ID NO:8)

[0239] LCDR2:KVS (SEQ ID NO:9)

[0240] LCDR3:FQGSHVPYT (SEQ ID NO: 10)

[0241] Amino acid sequence of 18H6H8:

[0242] The nucleotide sequence of 18H6H8 is:

[0243] Amino acid sequence of 18H6H14:

[0244] The nucleotide sequence of 18H6H14 is:

[0245] Amino acid sequence of 18H6H21:

[0246] The nucleotide sequence of 18H6H21 is:

[0247] Amino acid sequence of 18H6H23:

[0248] The nucleotide sequence of 18H6H23 is:

[0249] Amino acid sequence of 18H6H24:

[0250] The nucleotide sequence of 18H6H24 is:

[0251] Amino acid sequence of 18H6H25:

[0252] The nucleotide sequence of 18H6H25 is:

[0253] Amino acid sequence of 18H6H26:

[0254] The nucleotide sequence of 18H6H26 is:

[0255] Amino acid sequence of 18H6H27:

[0256] The nucleotide sequence of 18H6H27 is:

[0257] Amino acid sequence of 18H6H28:

[0258] The nucleotide sequence of 18H6H28 is:

[0259] Amino acid sequence of 18H6L3:

[0260] Nucleotide sequence of 18H6L3

[0261] Antibody heavy chain constant region amino acid sequence

[0262] Nucleotide sequence of the heavy chain constant region of the antibody

[0263] The amino acid sequence of the heavy chain constant region of the antibody (hG1TM)

[0264] Nucleotide sequence of the heavy chain constant region of antibody (hG1TM)

[0265] Amino acid sequence of the light chain constant region of hukappa

[0266] Nucleotide sequence of the light chain constant region of hukappa

[0267] Control antibody: CAN04

[0268] Amino acid sequence of CAN04-VH

[0269] Nucleotide sequence of CAN04-VH

[0270] Amino acid sequence of CAN04-VL

[0271] Nucleotide sequence of CAN04-VL

[0272] CAN04 heavy chain constant region amino acid sequence

[0273] CAN04 heavy chain constant region nucleotide sequence

[0274] Amino acid sequence of mFc:

Claims

1. An anti-IL-1RAP antibody or an antigen-binding fragment thereof, wherein the anti-IL-1RAP antibody comprises HCDR1 to HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 1, and LCDR1 to LCDR3 in the light chain variable region as shown in SEQ ID NO: 3, wherein preferably: according to the IMGT numbering system, The amino acid sequence of HCDR1 is shown in SEQ ID NO: 5, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 6, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 7, and The amino acid sequence of LCDR1 is shown in SEQ ID NO:8, the amino acid sequence of LCDR2 is KVS, shown in SEQ ID NO:9, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

10.

2. The anti-IL-1RAP antibody or antigen-binding fragment thereof according to claim 1, wherein; The anti-IL-1RAP antibody has a heavy chain variable region amino acid sequence comprising a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, and SEQ ID NO: 27, or a variant thereof; and The amino acid sequence of the light chain variable region of the anti-IL-1RAP antibody comprises a sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 29, or a variant 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.

3. The anti-IL-1RAP antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, wherein The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 3; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 29; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 29; The 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; The 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; The 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; The 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; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 23, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 29; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 25, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 29; or The 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.

4. The anti-IL-1RAP antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein The antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragment or single-chain antibody; the anti-IL-1RAP antibody is a humanized antibody or a chimeric antibody.

5. The anti-IL-1RAP antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein The anti-IL-1RAP antibody includes non-CDR regions, and the non-CDR regions are from a species other than murine, such as a human antibody, Preferably, the constant region of the anti-IL-1RAP antibody is selected from the constant region of human IgG1, IgG2, IgG3 or IgG4, Preferably, the anti-IL-1RAP antibody is of human IgG1 subtype.

6. The anti-IL-1RAP antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein The heavy chain constant region of the anti-IL-1RAP antibody adopts the Ig gamma-1 chain C region; the light chain constant region adopts the Ig kappa chain C region, or the heavy chain constant region amino acid sequence of the anti-IL-1RAP antibody is as shown in SEQ ID NO: 31 or 33, and the light chain constant region amino acid sequence is as shown in SEQ ID NO: 35, Preferably, according to the EU numbering system, based on the Ig gamma-1 chain C region (as shown in SEQ ID NO: 31), the heavy chain constant region of the anti-IL-1RAP antibody has the following mutations at positions 234, 235 and / or 237: L234A and L235A, L234A and G237A, L235A and G237A, or L234A, L235A, and G237A; Preferably, according to the EU numbering system, the heavy chain constant region of the antibody further has one or more mutations selected from the following: N297A, D265A, D270A, P238D, L328E, E233D, H268D, P271G, A330R, C226S, C229S, E233P, P331S, S267E, L328F, A330L, M252Y, S254T, T256E, N297Q, P238S, P238A, A327Q, A327G, P329A, K322A, T394D, G236R, G236A, L328R, A330S, H268A, E318A, and K320A, More preferably, the heavy chain constant region of the anti-IL-1RAP antibody is as shown in SEQ ID NO: 33; and the light chain constant region adopts the Ig kappa chain C region as shown in SEQ ID NO:

35.

7. The anti-IL-1RAP antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein EC binding of the anti-IL-1RAP antibody to cells expressing IL-1RAP 50 is less than or equal to 10 μg / mL, less than or equal to 8 μg / mL, less than or equal to 6 μg / mL, less than or equal to 4 μg / mL, or less than or equal to 3 μg / mL; preferably, the EC 50 It was measured by FACS (flow cytometry).

8. An isolated or synthetic polypeptide selected from the group consisting of: (1) An isolated or synthesized 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, and the antibody further comprises the sequences shown in SEQ ID NOs: 8, 9, and 10; (2) an isolated or synthesized polypeptide comprising the sequence 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 sequence shown in SEQ ID NOs: 5, 6, and 7; (3) an isolated or synthesized polypeptide comprising a sequence as shown in SEQ ID NO: 1, 11, 13, 15, 17, 19, 21, 23, 25 or 27, wherein the polypeptide specifically binds to IL-1RAP as part of an anti-IL-1RAP antibody, the antibody further comprising a sequence as shown in SEQ ID NO: 3 or 29; and (4) An isolated or synthesized 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.

9. A biomaterial selected from the group consisting of: (1) a nucleic acid molecule encoding the anti-IL-1RAP antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, or the isolated or synthesized polypeptide according to claim 8, (2) a recombinant vector comprising the nucleic acid molecule, and (3) A host cell comprising the nucleic acid molecule or the recombinant vector.

10. An antibody derivative comprising the anti-IL-1RAP antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, and selected from the group consisting of: (1) 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 (2) Fusion proteins or multispecific antibodies (such as bispecific antibodies).

11. A pharmaceutical composition comprising an effective amount of the anti-IL-1RAP antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, or the antibody derivative according to claim 10, optionally further comprising one or more pharmaceutically acceptable excipients.

12. The anti-IL-1RAP antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the antibody derivative according to claim 10, or the pharmaceutical composition according to claim 11, for use in treating tumors, inflammatory disorders, neurological adverse reactions, or autoimmune diseases.

13. Use of the anti-IL-1RAP antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, or the antibody derivative according to claim 10 in the preparation of a medicament for treating tumors, inflammatory diseases, neurological adverse reactions or autoimmune diseases.

14. A method for treating or preventing a tumor, comprising administering to a subject in need thereof an effective amount of the anti-IL-1RAP antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, the antibody derivative according to claim 10, or the pharmaceutical composition according to claim 11, preferably, the tumor is an IL-1RAP-positive tumor, preferably, the administration is before or after surgery, and / or before or after radiotherapy, preferably, the IL-1RAP-positive tumor is a solid tumor or a hematological tumor, preferably, the solid tumor is: prostate cancer, breast cancer ( The present invention relates to a group of cancers selected from the group consisting of 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, preferably, the hematological tumor is: multiple myeloma, myeloproliferative disorder (MPD), myelodysplastic syndrome (MDS), leukemia (chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML)).

15. 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 according to any one of claims 1 to 7, the antibody derivative according to claim 10, or the pharmaceutical composition according to claim 11, preferably, the autoimmune disease or inflammatory disease is an IL-1RAP-mediated condition. Preferably, the administration is before or after surgery, and / or before or after radiotherapy. More preferably, 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, irritable bowel syndrome, etc.

16. A method for treating or preventing an adverse neurological reaction, comprising administering to a subject in need thereof an effective amount of the anti-IL-1RAP antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, the antibody derivative according to claim 10, or the pharmaceutical composition according to claim 11, wherein the adverse neurological reaction is a central nervous system adverse reaction or a peripheral nervous system adverse reaction, and preferably, the peripheral nervous system adverse reaction is chemotherapy-induced peripheral neuropathy.

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