Anti-il1RAP antibody and use thereof

Abstract

A novel antibody and antibody fragment specifically binding to IL1RAP, and a composition containing the antibody or the antibody fragment. A nucleic acid encoding the antibody or the antibody fragment thereof, a host cell comprising the nucleic acid, and a related use. A therapeutic and diagnostic use of the antibody and the antibody fragment.

Description

Anti-IL1RAP antibodies and uses thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is based on the Chinese patent application with application number 202410396541.4 and application date April 2, 2024, and claims the priority of the Chinese patent application. The entire content of the Chinese patent application is hereby introduced into this application as a reference.

[0003] The present invention relates to novel antibodies and antibody fragments that specifically bind to IL1RAP, as well as compositions containing the antibodies or antibody fragments. Furthermore, the present invention relates to nucleic acids encoding the antibodies or antibody fragments thereof, host cells containing the same, and related uses. Furthermore, the present invention relates to therapeutic and diagnostic uses of these antibodies and antibody fragments.

[0004] Background of the Invention

[0005] Inhibition of inflammatory cytokine function is the cornerstone of many clinical interventions. Multiple members of the IL-1 family play important roles in a variety of diseases, such as IL-1B in gout, IL-33 in asthma, and IL-1α and IL-36 in psoriasis. Therefore, blocking specific disease-driving cytokines and inflammatory pathways may improve disease treatment outcomes.

[0006] The IL-1 family consists of 20 members, including 11 ligands and 9 receptors, and plays an important role in innate and adaptive immunity. Among them, IL-1R1 binds to IL-1α and IL-1β, recruiting IL-1R3 to form a trimeric signaling complex; IL-1R4 (ST2) binds to IL-33 and forms a trimeric complex with IL-1R3; and IL-1R6 binds to IL-36α, IL-36β, and IL-36γ, also recruiting IL-1R3 to form a trimeric complex. In the three different signaling pathways of IL1, IL33, and IL36, IL-1R3 acts as a co-receptor, forming receptor complexes with specific receptors for each pathway, thereby dimerizing the cytoplasmic Toll / IL-1R domain, triggering the activation of downstream signaling pathways and mediating specific immune responses.

[0007] IL-1R3, also known as interleukin-1 receptor accessory protein (IL1RAP), is a co-receptor for the type 1 interleukin-1 receptor (IL1R1) and is essential for IL-1 signaling. IL-1R3 mediates the signaling of six different cytokines and plays an important role in a variety of inflammatory and autoimmune diseases, including IL-1R3-dependent cytokine-induced inflammation, type 1 helper T cell (TH1) responses, type 2 helper T cell (TH2) responses, or a combination of inflammation with TH1 or TH2 responses. Therefore, blocking a common co-receptor, rather than a single primary receptor or ligand, may provide a new mode of action for reducing related diseases. Antibodies to IL1RAP can specifically bind to IL1RAP, thereby blocking IL1R1 from recruiting IL1RAP, and thereby inhibiting the activation of the NF-kB pathway by cytokines such as IL1.

[0008] The IL-1 pathway plays a crucial role in a variety of autoimmune and autoinflammatory diseases, including rheumatoid arthritis, gout, Still's disease, and cryopyrin-associated periodic syndrome (CAPS). Blocking the IL-1 pathway can effectively alleviate the symptoms of these diseases. Currently approved IL-1 inhibitors include the IL-1R antagonist Anakinra (Kineret), the IL-1B monoclonal antibody Canakinumab (Ilaris), and the IL-1 cytokine Trap, a fusion protein of the extracellular domains of IL-1R1 and IL-1RAP (Rilonacept (Arcalyst)). These drugs have demonstrated promising therapeutic effects in diseases such as rheumatoid arthritis, CAPS, and familial Mediterranean fever (FMF), and have been approved for marketing. However, these drugs all have various drawbacks. For example, Anakinra has a molecular weight of only 16.7 kd and a half-life of only 4-6 hours, necessitating daily dosing. Canakinumab only blocks IL-1B but not IL-1a, and therefore may not be effective for IL-1a-mediated diseases. Therefore, the present invention requires new antibodies targeting IL1RAP that can address the shortcomings of existing drugs.

[0009] SUMMARY OF THE INVENTION

[0010] The present invention provides a novel antibody that recognizes IL1RAP and selectively blocks a signaling pathway involved in IL1RAP.

[0011] In some embodiments, the present invention provides a novel monoclonal antibody targeting IL-1RAP, which has the characteristics of a long half-life of a monoclonal antibody and can simultaneously block IL-1a and 11-1B cytokines, thereby compensating for the shortcomings of known drugs in the prior art.

[0012] Therefore, the present invention relates to an antibody or fragment, such as an antigen-binding fragment, that specifically binds to IL1RAP, such as human IL1RAP. In some embodiments, the antibody or antigen-binding fragment thereof of the present invention binds to the K of human IL1RAP. D In some embodiments, the K is less than about 2 nM, 1.5 nM, 1.4 nM, 1.35 nM, 1.3 nM, 1.25 nM, 1.2 nM, 1.15 nM, 1.1 nM, 1.05 nM, 1 nM, 0.95 nM, 0.9 nM, 0.85 nM, 0.8 nM, 0.75 nM, 0.7 nM, 0.65 nM, 0.6 nM, 0.55 nM, 0.5 nM, 0.45 nM, or 0.4 nM, or greater than about 0.1 nM or 0.2 nM, or any range of values ​​therebetween. D Between 0.4 nM and 1.35 nM. In some embodiments, the affinity of antigen-antibody binding is detected by ForteBio.

[0013] The antibodies or antigen-binding fragments thereof of the present invention can effectively block the binding of IL1α, IL1β, IL33, IL36 to the IL1RAP receptor complex, thereby relieving the inhibitory effect on the downstream NF-kB signaling pathway. In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention can effectively block the activation of downstream signaling pathways mediated by IL1β. In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention effectively block the activation of downstream signaling pathways mediated by IL36. In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention effectively block the activation of downstream signaling pathways mediated by IL33. In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention effectively block the activation of downstream signaling pathways mediated by IL36, for example, in epithelial cells. In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention potently inhibit the IL-1, IL-36 and / or IL33 signaling pathways, for example, in PBMC cells.

[0014] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention have good pharmacokinetic characteristics in vivo, such as good stability and / or long half-life. In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention have a half-life of greater than about 300, 305, 310 or 315 hours, such as about 316 hours, when administered (e.g., by subcutaneous injection). In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention have a half-life of greater than about 175, 180, 185 or 190 hours, such as about 192 hours, when administered (e.g., by intravenous injection).

[0015] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention have good pharmacokinetic characteristics, such as good colloidal stability, good solubility, and / or good thermal stability.

[0016] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention are effective in preventing or treating inflammation, such as peritoneal inflammation such as peritonitis (e.g., acute peritonitis), gout (e.g., acute gout) and / or skin inflammation (e.g., dermatitis and psoriasis).

[0017] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention can effectively alleviate peritoneal inflammation, such as reducing the recruitment of inflammatory cells in the peritoneal cavity and / or reducing the number of infiltrating neutrophils and / or monocytes.

[0018] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention can effectively alleviate the onset of gout and / or inhibit paw thickening, and optionally also reduce the production of inflammatory factors in paw tissue.

[0019] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention are effective in inhibiting skin inflammation (eg, dermatitis), such as reducing skin thickness.

[0020] Therefore, the present invention relates to the following specific aspects.

[0021] In some aspects, the present invention relates to an anti-IL1RAP antibody or an antigen-binding fragment thereof, comprising:

[0022] The three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in any one of SEQ ID NOs: 13-17, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in any one of SEQ ID NOs: 23-25.

[0023] In some aspects, the present invention relates to an anti-IL1RAP antibody or an antigen-binding fragment thereof, comprising a first heavy chain complementarity determining region (HCDR1), a second heavy chain complementarity determining region (HCDR2), a third heavy chain complementarity determining region (HCDR3) and a first light chain complementarity determining region (LCDR1), a second light chain complementarity determining region (LCDR2) and a third light chain complementarity determining region (LCDR3), wherein the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise or consist of the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

[0024] In some embodiments, the anti-IL1RAP antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH), wherein the heavy chain variable region comprises, consists of, or comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 13-17, or consists of said sequence.

[0025] In some embodiments, the anti-IL1RAP antibody or antigen-binding fragment thereof of the present invention comprises a light chain variable region (VL), wherein the light chain variable region comprises, consists of, or comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 23-25, or consists of said sequence.

[0026] In some embodiments, the anti-IL1RAP antibody or antigen-binding fragment thereof of the present invention comprises

[0027] (i) a VH comprising, or consisting of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to, or consisting of, the amino acid sequence of SEQ ID NO: 13, and a VL comprising, or consisting of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to, or consisting of, the amino acid sequence of SEQ ID NO: 23;

[0028] (ii) a VH comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 14 or 15, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and a VL comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 24, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0029] (iii) a VH comprising, or consisting of, an amino acid sequence represented by SEQ ID NO: 16 or 17, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and a VL comprising, or consisting of, an amino acid sequence represented by SEQ ID NO: 25, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0030] In some embodiments, the anti-IL1RAP antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region, wherein

[0031] (i) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16 or 17, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 25;

[0032] (ii) the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 16 or 17, and the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 25;

[0033] (iii) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 13, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 23;

[0034] (iv) the heavy chain variable region consists of the amino acid sequence set forth in SEQ ID NO: 13, and the light chain variable region consists of the amino acid sequence set forth in SEQ ID NO: 23;

[0035] (v) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 14 or 15, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 24; or

[0036] (vi) the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 14 or 15, and the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 24.

[0037] In some embodiments, an anti-IL1RAP antibody or antigen-binding fragment thereof of the invention comprises an Fc region, eg, an Fc region from IgG1, IgG2, IgG3, or IgG4, eg, a human IgG1, IgG2, IgG3, or IgG4.

[0038] In some embodiments, the anti-IL1RAP antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain constant region derived from a constant region of IgG1, IgG2, IgG3, or IgG4, such as a constant region of human IgG1, IgG2, IgG3, or IgG4, such as the IgG1 heavy chain constant region.

[0039] (i) comprises or consists of the amino acid sequence of SEQ ID NO: 50; or

[0040] (ii) comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:50.

[0041] In some embodiments, the heavy chain constant region or Fc region of the present invention lacks a C-terminal lysine. In some embodiments, the heavy chain constant region or Fc region of the present invention comprises a mutation that reduces binding to Fcγ receptors, such as an L234A / L235A mutation and / or a P329G mutation, such as an L234A / L235A mutation or an L234A / L235A mutation and a P329G mutation.

[0042] In some embodiments, the heavy chain constant region

[0043] (i) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 51 or 52;

[0044] (ii) comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 51 and has an L234A / L235A mutation; or

[0045] (iii) comprising an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 52, and having an L234A / L235A mutation and a P329G mutation.

[0046] In some embodiments, the anti-IL1RAP antibody or antigen-binding fragment thereof of the present invention comprises a light chain constant region, wherein the light chain constant region is a Lambda or Kappa light chain constant region, such as a human Lambda or Kappa light chain constant region. In some embodiments, the light chain constant region

[0047] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 53; or

[0048] (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 53.

[0049] In some embodiments, the anti-IL1RAP antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain, wherein the heavy chain

[0050] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 32-36; or

[0051] (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 32-36.

[0052] In some embodiments, the anti-IL1RAP antibodies or antigen-binding fragments thereof of the present invention comprise a light chain, wherein the light chain

[0053] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 42-44; or

[0054] (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 42-44.

[0055] In some embodiments, the anti-IL1RAP antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and a light chain, wherein

[0056] (i) the heavy chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:32, and the light chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:42;

[0057] (ii) the heavy chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 33 or 34, and the light chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 43; or

[0058] (iii) the heavy chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 35 or 36, and the light chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 44.

[0059] In some embodiments, the anti-IL1RAP antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and a light chain, wherein

[0060] (i) the heavy chain comprises the amino acid sequence of SEQ ID NO: 35 or 36, and the light chain comprises the amino acid sequence of SEQ ID NO: 44;

[0061] (ii) the heavy chain consists of the amino acid sequence of SEQ ID NO: 35 or 36, and the light chain consists of the amino acid sequence of SEQ ID NO: 44;

[0062] (iii) the heavy chain comprises the amino acid sequence of SEQ ID NO: 32, and the light chain comprises the amino acid sequence of SEQ ID NO: 42;

[0063] (iv) the heavy chain consists of the amino acid sequence of SEQ ID NO: 32, and the light chain consists of the amino acid sequence of SEQ ID NO: 42;

[0064] (v) the heavy chain comprises the amino acid sequence of SEQ ID NO: 33 or 34, and the light chain comprises the amino acid sequence of SEQ ID NO: 43; or

[0065] (vi) the heavy chain consists of the amino acid sequence of SEQ ID NO: 33 or 34, and the light chain consists of the amino acid sequence of SEQ ID NO: 43.

[0066] In some embodiments, the anti-IL1 RAP antibodies of the invention are monoclonal antibodies.

[0067] In some embodiments, the anti-IL1RAP antibodies of the invention are humanized antibodies or chimeric antibodies.

[0068] In some embodiments, the antigen-binding fragment of the anti-IL1RAP antibody of the present invention is an antibody fragment selected from the group consisting of: Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, single-domain antibody such as VHH, dAb (domain antibody), diabody, or linear antibody.

[0069] In some aspects, the invention relates to an isolated nucleic acid encoding an anti-IL1 RAP antibody or antigen-binding fragment thereof of the invention.

[0070] In some aspects, the present invention relates to a vector comprising the nucleic acid of the present invention, preferably the vector is an expression vector.

[0071] In some aspects, the present invention relates to a host cell comprising a nucleic acid or vector of the present invention, preferably, the host cell is prokaryotic or eukaryotic, more preferably selected from yeast cells, mammalian cells (such as 293 cells or CHO cells, such as CHO-K cells or HEK293 cells) or other cells suitable for preparing antibodies or antigen-binding fragments thereof.

[0072] In some aspects, the present invention relates to a method for preparing an anti-IL1RAP antibody or an antigen-binding fragment thereof, the method comprising

[0073] a) culturing the host cell of the present invention under conditions suitable for expressing a nucleic acid encoding an anti-IL1RAP antibody or antigen-binding fragment thereof of the present invention,

[0074] b) optionally isolating said antibody or antigen-binding fragment thereof,

[0075] c) Optionally, the method further comprises recovering the anti-IL1RAP antibody or antigen-binding fragment thereof from the host cell. Optionally, the antibody is purified, for example, by Protein A purification.

[0076] In some aspects, the invention relates to an immunoconjugate comprising an anti-IL1RAP antibody of the invention or an antigen-binding fragment thereof and other substances, such as chemotherapeutic agents, toxins, small molecule drugs, cytotoxic agents, apoptotic agents, chelating agents, immunomodulatory agents, such as immunosuppressive agents.

[0077] In some aspects, the present invention relates to a pharmaceutical composition comprising the anti-IL1RAP antibody or antigen-binding fragment or immunoconjugate of the present invention, and optionally a pharmaceutically acceptable excipient.

[0078] In some aspects, the invention relates to a pharmaceutical combination product comprising an anti-IL1RAP antibody of the invention or an antigen-binding fragment or immunoconjugate thereof, and one or more other therapeutic agents, e.g., the therapeutic agent is suitable for treating inflammation, e.g., selected from cytokines, other antibodies, small molecule drugs or immunomodulators (e.g., immunosuppressants).

[0079] In some aspects, the present invention relates to a method for preventing or treating a disease or condition associated with inappropriate activation of a pathway mediated by an IL1RAP receptor complex in an individual, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof, or immunoconjugate, or pharmaceutical composition, or pharmaceutical combination product of the present invention that binds to IL1RAP. In some embodiments, the subject has abnormal activation of an IL1RAP receptor complex-mediated signaling pathway compared to a healthy individual. In some embodiments, the disease or condition is selected from a tumor, such as cancer, or an inflammation, such as peritoneal inflammation, such as peritonitis (e.g., acute peritonitis), gout (e.g., acute gout), and / or skin inflammation (e.g., dermatitis).

[0080] In some embodiments, the method further comprises administering one or more other therapies, e.g., treatment modalities and / or other therapeutic agents, e.g., agents useful for treating inflammation, e.g., selected from cytokines, other antibodies, small molecule drugs, or immunomodulators (e.g., immunosuppressants). Description of the drawings:

[0081] Figure 1 shows the binding of chimeric and humanized antibodies to CHO-hIL1RAP cells.

[0082] FIG2 shows the inhibitory activity of humanized antibodies in an IL-1 fluorescent reporter cell line.

[0083] FIG3 shows the inhibitory activity of humanized antibodies in an IL-36 fluorescent reporter cell line.

[0084] FIG4 shows the inhibitory activity of humanized antibodies in an IL-33 fluorescent reporter cell line.

[0085] FIG5 shows the inhibitory activity of chimeric antibodies and humanized antibodies on the IL36 pathway in the human squamous cell carcinoma cell line A431.

[0086] FIG6 shows the inhibitory activity of chimeric and humanized antibodies on the IL1 pathway in PBMCs.

[0087] FIG7 shows the inhibitory activity of chimeric and humanized antibodies on the IL36 pathway in PBMCs.

[0088] FIG8 shows the inhibitory activity of chimeric and humanized antibodies on the IL33 pathway in PBMCs.

[0089] FIG9 shows the results of validating the biological activity of the antibody on the IL1 pathway in a mouse acute peritonitis model.

[0090] FIG10 shows the results of validating the biological activity of the antibody on the IL1 pathway in a mouse acute gout model.

[0091] FIG11 shows the biological activity of antibodies against the IL36 pathway validated in a mouse ear dermatitis model.

[0092] Detailed Description of the Invention

[0093] Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described herein as these may vary.

[0094] I. Definition

[0095] It should be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0096] To interpret this specification, the following definitions will apply, and wherever appropriate, terms used in the singular may also include the plural, and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0097] The term "about" when used in conjunction with a numerical value is meant to encompass a range of numerical values ​​having a lower limit that is 5% (e.g., 4%, 3%, 2% or 1%) less than the specified numerical value and an upper limit that is 5% (e.g., 4%, 3%, 2% or 1%) greater than the specified numerical value.

[0098] As used herein, the term "and / or" means any one of the alternatives or two or more or all of the alternatives.

[0099] As used herein, the terms "comprising" or "including" are intended to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms "comprising" or "including" are used, unless otherwise indicated, combinations / consistencies of the stated elements, integers, or steps are also encompassed. For example, when reference is made to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of the specific sequence.

[0100] An "isolated" antibody or molecule is one that has been separated from a component of its natural environment. In some embodiments, the antibody or molecule is purified to greater than 95% or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC).

[0101] As used herein, "IL1RAP" refers to any native IL1RAP polypeptide (e.g., a human IL1RAP polypeptide) or a variant thereof. The term "IL1RAP" encompasses "full-length" unprocessed IL1RAP polypeptides as well as any form of IL1RAP polypeptides produced by processing within a cell. The term also encompasses naturally occurring variants of IL1RAP, such as those encoded by splice variants and allelic variants. The IL1RAP polypeptides described herein can be isolated from a variety of sources, such as from humans or from another source, such as cynomolgus monkeys, or prepared by recombinant or synthetic methods. In one embodiment of the invention, the human IL1RAP protein is as shown in Uniprot, Q9NPH3. In one embodiment, the human IL1RAP gene is as shown in NCBI Gene ID: 3556. In one embodiment of the invention, the cynomolgus monkey IL1RAP protein is as shown in Uniprot, G7NYP7.

[0102] The terms "whole antibody" or "full-length antibody" are used interchangeably herein and refer to antibody molecules with natural immunoglobulin molecular structure. In the case of conventional four-chain IgG antibodies, the full-length antibody comprises two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. In the case of heavy chain antibodies having only heavy chains and lacking light chains, the full-length antibody comprises two heavy chains (H) interconnected by disulfide bonds. For conventional four-chain IgG antibodies, the full-length antibody heavy chain is generally composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region, wherein the heavy chain constant region at least comprises three domains CH1, CH2 and CH3 (optionally also comprising CH4). The full-length antibody light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region, wherein the light chain constant region consists of one domain CL. Each heavy chain variable region (VH) and each light chain variable region (VH) is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term "antibody fragment" includes a portion of an intact antibody. In preferred embodiments, the antibody fragment is an antigen-binding fragment.

[0103] The term "antigen-binding fragment" of an antibody is a molecule that is different from a full-length antibody and that contains a portion of a full-length antibody, but that can bind to the antigen of the full-length antibody or compete with the full-length antibody (i.e., the full-length antibody from which the antigen-binding fragment is derived) for antigen binding. Antigen-binding fragments can be prepared by recombinant DNA technology, or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, dAb (domain antibody), linear antibodies, single-chain antibodies (e.g., scFv); single-domain antibodies such as VHH, diabodies or fragments thereof, or camelid antibodies, diabodies, single-domain antibodies (sdAb), and nanobodies. For example, Fab fragments can be obtained by digesting a full-length antibody with papain. In addition, digesting a full antibody below the disulfide bonds in the hinge region with pepsin produces F(ab')2, which is a dimer of Fab' and a divalent antibody fragment. F(ab')2 can be reduced under neutral conditions by destroying the disulfide bonds in the hinge region, thereby converting the F(ab')2 dimer into a Fab' monomer. A Fab' monomer is essentially a Fab fragment with a hinge region. An Fv fragment consists of the VL and VH domains of a single antibody arm. The two domains of the Fv fragment, VL and VH, can be encoded by separate genes, but recombinant methods can also be used to connect the two domains using a synthetic linker peptide to produce them as a single protein chain, in which the VL and VH regions are paired to form a single-chain Fv (scFv).

[0104] The term "single-chain antibody (scAb)" is used herein in the broadest sense and specifically covers antibodies with monospecificity or multispecificity (e.g., bispecificity) that are initially produced as a single continuous polypeptide chain. Such single-chain antibodies include, but are not limited to, antibodies having two linked VL and VH regions. In one embodiment, the single-chain antibody is an scFv.

[0105] "Diabodies" are small, bivalent antibodies constructed through gene fusion, for example, dimers composed of two polypeptide chains. The VL and VH domains of each polypeptide chain of a diabody are linked by a linker, so that the VL and VH encoded in the same polypeptide chain form a dimer with different single-chain variable region segments. Diabodies generally have two antigen-binding sites.

[0106] "Complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is highly variable in sequence and forms structurally determined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, and are numbered sequentially starting from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment schemes, including, for example, Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (www.imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0107] The following are exemplary schemes for the regional extent of CDRs defined using the Kabat, AbM, Chothia, Contact, and IMGT schemes.

[0108] Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above ways or in combination. CDRs can also be determined based on having the same Kabat numbering position as a reference CDR sequence (e.g., any of the exemplary CDRs of the present invention).

[0109] Unless otherwise indicated, in the present invention, when referring to residue positions in the variable region of an antibody (including heavy chain variable region residues and light chain variable region residues), the numbering refers to the position according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0110] In some embodiments, the CDR1 of the heavy chain variable region of the antibody of the present invention is determined according to the Kabat & Chothia scheme, and the CDR2 and CDR3 are determined according to the Kabat scheme. In some embodiments, the CDRs of the light chain variable region of the antibody of the present invention are determined according to the Kabat scheme. In some embodiments, the CDRs of the light chain variable region of the antibody of the present invention are determined according to the Kabat scheme.

[0111] HCDR1 was determined according to the Kabat & Chothia scheme, and HCDR2 and HCDR3 were determined according to the Kabat scheme;

[0112] LCDR1, LCDR2 and LCDR3 were determined according to the Kabat protocol.

[0113] An "antibody that binds to the same or overlapping epitope as a reference antibody" is an antibody that blocks 50%, 60%, 70%, 80%, 90% or 95% or more of the binding of the reference antibody to its antigen in a competition assay, whereas conversely, the reference antibody blocks 50%, 60%, 70%, 80%, 90% or 95% or more of the binding of the antibody to its antigen in a competition assay.

[0114] An antibody that competes with a reference antibody for binding to its antigen is an antibody that blocks 50%, 60%, 70%, 80%, 90% or 95% or more of the binding of the reference antibody to its antigen in a competition assay. Conversely, a reference antibody blocks 50%, 60%, 70%, 80%, 90% or 95% or more of the binding of the antibody to its antigen in a competition assay. Numerous types of competitive binding assays can be used to determine whether one antibody competes with another, such as solid phase direct or indirect radioimmunoassays (RIAs), solid phase direct or indirect enzyme immunoassays (EIAs), and sandwich competition assays.

[0115] An antibody that inhibits (e.g., competitively inhibits) the binding of a reference antibody to its antigen is an antibody that inhibits the binding of the reference antibody to its antigen by 50%, 60%, 70%, 80%, 90%, or 95% or more. Conversely, the reference antibody inhibits the binding of the antibody to its antigen by 50%, 60%, 70%, 80%, 90%, or 95% or more. The binding of an antibody to its antigen can be measured by affinity (e.g., equilibrium dissociation constant). Methods for determining affinity are known in the art.

[0116] An antibody that exhibits the same or similar binding affinity and / or specificity as a reference antibody is an antibody that has at least 50%, 60%, 70%, 80%, 90% or more than 95% of the binding affinity and / or specificity of the reference antibody. This can be determined by any method known in the art for determining binding affinity and / or specificity.

[0117] The term "chimeric antibody" is an antibody molecule in which (a) the constant region or a portion thereof is changed, replaced or exchanged so that the antigen binding site is connected to a constant region of a different or altered class, effector function and / or species or a completely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug) that imparts new properties to the chimeric antibody; or (b) the variable region or a portion thereof is changed, replaced or exchanged with a variable region having a different or altered antigenic specificity. For example, a mouse antibody can be modified by replacing its constant region with a constant region from a human immunoglobulin. Due to the replacement with a human constant region, the chimeric antibody can retain its specificity in recognizing the antigen while having reduced immunogenicity in humans as compared to the original mouse antibody.

[0118] A "humanized antibody" is an antibody that retains the antigen-specific reactivity of a non-human antibody (e.g., a mouse monoclonal antibody) while being less immunogenic when administered to humans, for example, as a therapeutic. This can be achieved, for example, by retaining the non-human antigen-binding site and replacing the remaining portions of the antibody with their human counterparts (i.e., replacing the constant region and portions of the variable region not involved in binding with the corresponding portions of a human antibody).

[0119] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" comprises two or three constant domains, namely a CH2 domain, a CH3 domain, and an optional CH4 domain. For example, in a native antibody, an immunoglobulin Fc domain comprises the second and third constant domains (CH2 domain and CH3 domain) of two heavy chains derived from IgG, IgA, and IgD class antibodies; or the second, third, and fourth constant domains (CH2 domain, CH3 domain, and CH4 domain) of two heavy chains derived from IgM and IgE class antibodies. Unless otherwise indicated herein, amino acid residue numbering in the Fc region or heavy chain constant region is according to the EU numbering system (also called the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0120] As used herein, "conservative changes" include substitutions, deletions, or additions to a polypeptide sequence that do not substantially alter the desired functional activity of the polypeptide sequence. In some embodiments, conservative changes are conservative substitutions. A conservative substitution refers to the replacement of one amino acid with another within the same class, such as the replacement of an acidic amino acid with another acidic amino acid, the replacement of a basic amino acid with another basic amino acid, or the replacement of a neutral amino acid with another neutral amino acid. For example, conservative substitutions often result in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following lists 8 groups of amino acids containing conservative substitutions for each other: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine ​​(C), Methionine (M). In some embodiments, the term "conservative change" when applied to an antibody molecule amino acid sequence refers to an amino acid modification that does not significantly affect or change the target antigen binding characteristics of the antibody molecule of the present invention containing the amino acid sequence. For example, a conservatively changed variant maintains at least 80%, 85%, 90%, 95%, 98%, 99% or higher, such as 100-110% or higher, binding affinity for the target antigen relative to the parent antibody.

[0121] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0122] An "immunoconjugate" is an antibody conjugated to one or more other substances, including but not limited to a label. In some embodiments, the immunoconjugate encompasses an antibody-drug conjugate (ADC).

[0123] The term "therapeutic agent" as used herein encompasses any substance effective in preventing or treating diseases associated with inappropriate activation of pathways mediated by the IL1RAP receptor complex, including cytokines, other antibodies, small molecule drugs, or immunomodulators (eg, immunosuppressants).

[0124] The term "small molecule drug" refers to low molecular weight organic compounds that are capable of regulating biological processes. "Small molecules" are defined as molecules with a molecular weight of less than 10 kD, typically less than 2 kD, and preferably less than 10 kD. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimics. As therapeutic agents, small molecules can be more cell-permeable, less susceptible to degradation, and less prone to eliciting an immune response than macromolecules.

[0125] As used herein, the term "immunomodulator" refers to a natural or synthetic agent or drug that inhibits or modulates an immune response. The immune response can be a humoral response or a cellular response. Immunomodulators include immunosuppressants.

[0126] As used herein, an "immunosuppressant," "immunosuppressive drug," or "immunosuppressant" is a therapeutic agent used in immunosuppressive therapy to suppress or prevent the activity of the immune system.

[0127] The term "effective amount" refers to an amount or dosage of an antibody or fragment or conjugate or composition or combination of the present invention that produces the desired effect in a patient in need of treatment or prevention after administration in single or multiple doses to the patient.

[0128] A "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic outcome at the required dosage and for the required period of time. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or its conjugate or composition or combination are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., inhibition of the IL-1 signaling pathway) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60%, or 70% relative to an untreated subject. In some embodiments, the term "therapeutically effective amount" as used herein is intended to define the amount of treatment necessary to treat a condition (e.g., inflammation) or to reduce or eliminate an immune response in a therapeutic regimen.

[0129] A "prophylactically effective amount" refers to an amount effective to achieve the desired preventive result at the required dosage and for the required period of time. Typically, because a prophylactic dose is used in a subject before or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount. In some embodiments, the term "prophylactically effective amount" as used herein is intended to define the amount necessary to prevent the progression and symptoms (e.g., inflammation) of a condition or disease in a treatment regimen.

[0130] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny screened or selected for the same function or biological activity as the initially transformed cell are included herein.

[0131] The term "label" as used herein refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as a polynucleotide probe or antibody) and promotes the detection of the reagent to which it is conjugated or fused. The label itself can be detectable (e.g., a radioisotope label or a fluorescent label) or can catalyze a chemical change in a detectable substrate compound or composition in the case of an enzymatic label. The term is intended to encompass direct labeling of a probe or antibody by coupling (i.e., physically connecting) a detectable substance to the probe or antibody and indirect labeling of the probe or antibody by reacting with another reagent of the direct label.

[0132] As used herein, the terms "individual," "subject," or "subject" are used interchangeably and include mammals. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0133] As used herein, the term "subject / patient / individual sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of a tissue or cell sample can be solid tissue, such as an organ or tissue sample or a biopsy sample or a puncture sample from fresh, frozen and / or preserved; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time during the subject's pregnancy or development. Tissue samples may contain compounds that are not naturally contaminated with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.

[0134] "Nucleic acid encoding an anti-IL1RAP antibody or a fragment thereof" refers to one or more nucleic acid molecules that encode an antibody heavy chain or light chain (or a fragment thereof, such as a heavy chain variable region or a light chain variable region), including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present at one or more locations in a host cell.

[0135] "Percent identity (%)" of an amino acid sequence refers to the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the specific amino acid sequence set forth in this specification, after aligning the candidate sequence with the specific amino acid sequence set forth in this specification and introducing gaps, if necessary, to achieve the maximum percentage identity, and not considering any conservative substitutions as part of the sequence identity. In some embodiments, the present invention contemplates variants of the antibody molecules of the present invention that have a substantial degree of identity, e.g., at least 80%, 85%, 90%, 95%, 97%, 98% or 99% or more, relative to the antibody molecules and sequences thereof specifically disclosed herein. Such variants may comprise conservative changes.

[0136] The term "pharmaceutical excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, or the like, which is administered together with the active substance.

[0137] The term "pharmaceutical composition" refers to a composition that is in form permitting the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0138] As used herein, the term "drug combination" or "combination product" refers to a non-fixed combination or a fixed combination, including but not limited to a kit / test kit, a pharmaceutical composition. The term "non-fixed combination" means that the active ingredients (e.g., (i) an antibody of the present invention, and (ii) other therapeutic agent) are administered to a patient simultaneously, without specific time restrictions, or sequentially at the same or different time intervals, as separate entities, wherein such administration provides two or more active agents with prophylactic or therapeutically effective levels in the patient's body. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. The dosage and / or time interval of the two or more active agents are preferably selected so that the combined use of the parts can produce an effect greater than that achieved by using any one component alone when treating a disease or condition. Each component can be in the form of a separate formulation, which can be the same or different.

[0139] The term "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities (e.g., radiotherapy or surgery) to treat diseases described herein. This administration includes co-administering these therapeutic agents in a substantially simultaneous manner, such as in a single capsule with a fixed ratio of active ingredients. Alternatively, this administration includes co-administration of each active ingredient in a variety of or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids can be reconstituted or diluted to the desired dose before administration. In addition, this administration also includes using each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the therapeutic regimen will provide the beneficial effects of the drug combination in treating disorders or conditions described herein.

[0140] As used herein, "treat," ...

[0141] As used herein, "prevention" includes the inhibition of the onset or development of a disease or condition or symptoms of a particular disease or condition.

[0142] II Antibodies

[0143] In some embodiments, the anti-IL1RAP antibodies or antigen-binding fragments thereof of the present invention comprise three complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3.

[0144] In some embodiments, the anti-IL1RAP antibodies or antigen-binding fragments thereof of the present invention comprise three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.

[0145] In some embodiments, an anti-IL1RAP antibody or antigen-binding fragment thereof of the present invention comprises three complementarity determining regions (HCDRs) from a heavy chain variable region and three complementarity determining regions (LCDRs) from a light chain variable region.

[0146] In some aspects, the anti-IL1RAP antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region (VH). In some aspects, the anti-IL1RAP antibodies or antigen-binding fragments thereof of the present invention comprise a light chain variable region (VH). In some aspects, the anti-IL1RAP antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region and a light chain variable region (VH). In some embodiments, the heavy chain variable region comprises three complementary determining regions (CDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises three complementary determining regions (CDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.

[0147] In some embodiments, the anti-IL1RAP antibodies or antigen-binding fragments thereof of the present invention further comprise an antibody heavy chain constant region. In some embodiments, the anti-IL1RAP antibodies or antigen-binding fragments thereof of the present invention further comprise an antibody light chain constant region. In some embodiments, the anti-IL1RAP antibodies or antigen-binding fragments thereof of the present invention further comprise a heavy chain constant region and a light chain constant region.

[0148] In some embodiments, the heavy chain variable region:

[0149] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 13-17; or

[0150] (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 13-17; or

[0151] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in any one of SEQ ID NOs: 13-17, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions.

[0152] In some embodiments, the light chain variable region

[0153] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 23-25; or

[0154] (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 23-25; or

[0155] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in any one of SEQ ID NOs: 23-25, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions.

[0156] In some embodiments, the three complementarity determining regions (HCDRs) from the heavy chain variable region of the present invention, HCDR1, HCDR2 and HCDR3 are selected from

[0157] (i) three complementarity determining regions HCDR1, HCDR2, and HCDR3 contained in the VH as shown in any one of SEQ ID NOs: 13-17;

[0158] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably an amino acid substitution, preferably a conservative substitution) in the three HCDR regions relative to the sequence of any one of (i),

[0159] wherein the HCDRs can be determined according to any scheme for determining CDRs, for example, according to the Kabat, AbM, Chothia, Contact or IMGT schemes or a combination thereof;

[0160] For example, the HCDR1 is determined according to the Kabat & Chothia scheme, and the HCDR2 and HCDR3 are each determined according to the Kabat scheme.

[0161] In some embodiments, the three complementarity determining regions (LCDRs) from the light chain variable region of the present invention, LCDR1, LCDR2 and LCDR3 are selected from

[0162] (i) three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in any one of SEQ ID NOs: 23-25, or

[0163] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably an amino acid substitution, preferably a conservative substitution) in the three LCDR regions relative to the sequence of any one of (i),

[0164] wherein the HCDRs can be determined according to any scheme for determining CDRs, for example, according to the Kabat, AbM, Chothia, Contact or IMGT schemes or a combination thereof;

[0165] For example, the LCDRs 1, 2 and 3 are each determined according to the Kabat protocol.

[0166] In some embodiments, the anti-IL1RAP antibody or antigen-binding fragment thereof of the present invention comprises:

[0167] (i) three complementarity determining regions HCDR1, HCDR2, and HCDR3 contained in the VH as shown in any one of SEQ ID NOs: 13-17, and three complementarity determining regions LCDR1, LCDR2, and LCDR3 contained in the VL as shown in any one of SEQ ID NOs: 23-25;

[0168] (ii) three complementarity determining regions HCDR1, HCDR2, and HCDR3 contained in the VH as shown in SEQ ID NO: 13, and three complementarity determining regions LCDR1, LCDR2, and LCDR3 contained in the VL as shown in SEQ ID NO: 23;

[0169] (iii) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 14 or 15, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: 24; or

[0170] (iv) three complementarity determining regions HCDR1, HCDR2, and HCDR3 contained in the VH as shown in SEQ ID NO: 16 or 17, and three complementarity determining regions LCDR1, LCDR2, and LCDR3 contained in the VL as shown in SEQ ID NO: 25;

[0171] wherein the HCDRs can be determined according to any scheme for determining CDRs, for example, according to the Kabat, AbM, Chothia, Contact or IMGT schemes or a combination thereof;

[0172] For example, the HCDR1 is determined according to the Kabat & Chothia scheme, and the HCDR2 and HCDR3 are each determined according to the Kabat scheme.

[0173] In some embodiments, the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, or consists of the amino acid sequence, or the HCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 1.

[0174] In some embodiments, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, or consists of the amino acid sequence, or the HCDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 2.

[0175] In some embodiments, the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3, or the HCDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 3.

[0176] In some embodiments, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, or consists of the amino acid sequence, or LCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 4.

[0177] In some embodiments, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, or consists of the amino acid sequence, or LCDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 5.

[0178] In some embodiments, the LCDR3 comprises the amino acid sequence of SEQ ID NO: 6, or consists of the amino acid sequence, or the LCDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 6.

[0179] In some embodiments, the antibody heavy chain constant region of the present invention is a heavy chain constant region from IgG1, IgG2, IgG3 or IgG4, such as a constant region of human IgG1, IgG2, IgG3 or IgG4, such as a constant region of human IgG1, IgG2, IgG3 or IgG4. In some embodiments, the constant region has a modification in the Fc region. In one embodiment, the Fc region is modified on the characteristics of the effector function (such as the complement activation function of the Fc region) in the Fc region. In one embodiment, the effector function has been reduced or eliminated relative to the wild isotype Fc region. In one embodiment, the effector function is reduced or eliminated by a method selected from the following: using an Fc isotype and Fc region modification with a natural effector function that is reduced or eliminated. The Fc region can also include modifications that change the binding affinity to one or more Fc receptors. In one embodiment, the Fc receptor is an Fc γ receptor, particularly a human Fc γ receptor. In some embodiments, the Fc region includes a mutation that reduces binding to the Fc γ receptor. In some embodiments, the Fc region used in the present invention has L234A / L235A mutations and / or P329G mutations that reduce binding to Fcγ receptors.

[0180] In some embodiments, the heavy chain constant region of the present invention comprises a mutation that reduces binding to Fcγ receptors, such as L234A / L235A mutation and / or P329G mutation, such as L234A / L235A mutation or L234A / L235A mutation and P329G mutation. In some embodiments, the heavy chain constant region

[0181] (i) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 50, 51 or 52;

[0182] (ii) comprising an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 50, 51 or 52

[0183] (iii) comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 51 and has an L234A / L235A mutation; or

[0184] (iv) comprising an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 52, and having an L234A / L235A mutation and a P329G mutation.

[0185] In some embodiments, the heavy chain constant regions of the invention lack a C-terminal lysine.

[0186] In some embodiments, the antibody light chain constant region of the present invention is derived from a Lambda or Kappa light chain constant region, preferably a Kappa light chain constant region, such as a human Lambda or Kappa light chain constant region, such as a human Lambda or Kappa light chain constant region. In some embodiments, the light chain constant region

[0187] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 53; or

[0188] (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 53.

[0189] In some specific embodiments, the anti-IL1RAP antibody of the present invention comprises a first heavy chain complementarity determining region (HCDR1), a second heavy chain complementarity determining region (HCDR2), a third heavy chain complementarity determining region (HCDR3) and a first light chain complementarity determining region (LCDR1), a second light chain complementarity determining region (LCDR2) and a third light chain complementarity determining region (LCDR3), wherein the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise or consist of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

[0190] In some embodiments, the VH of the present invention comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, and the HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, or consist of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and the LCDR1, LCDR2, and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, or consist of the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0191] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises:

[0192] (i) a VH comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 13, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and / or a VL comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 23, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0193] (ii) a VH comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 14 or 15, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and / or a VL comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 24, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0194] (iii) a VH comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 16 or 17, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and / or a VL comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 25, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0195] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region and a light chain variable region, wherein

[0196] (i) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 13, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 23;

[0197] (ii) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 14 or 15, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 24; or

[0198] (iii) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 16 or 17, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 25.

[0199] In some embodiments, the anti-IL1RAP antibodies or antigen-binding fragments thereof of the present invention comprise an antibody heavy chain. In some embodiments, the anti-IL1RAP antibodies or antigen-binding fragments thereof of the present invention comprise an antibody light chain. In some embodiments, the anti-IL1RAP antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain and a light chain. In some embodiments, the heavy chain of the antibody of the present invention comprises the heavy chain variable region and the heavy chain constant region, or consists of the heavy chain variable region and the heavy chain constant region. In some embodiments, the light chain of the antibody of the present invention comprises the light chain variable region and the light chain constant region, or consists of the light chain variable region and the light chain constant region. In some embodiments, the antibodies of the present invention comprise two heavy chains and two light chains, or consist of two heavy chains and two light chains.

[0200] In some embodiments, the heavy chain comprises

[0201] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 32-36;

[0202] (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 32-36; or

[0203] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from any one of SEQ ID NOs: 32-36.

[0204] In some embodiments, the light chain comprises

[0205] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 42-44;

[0206] (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 42-44; or

[0207] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from any one of SEQ ID NOs: 42-44.

[0208] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain and a light chain, wherein

[0209] (i) the heavy chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:32, and the light chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:42;

[0210] (ii) the heavy chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 33 or 34, and the light chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 43; or

[0211] (iii) the heavy chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 35 or 36, and the light chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 44.

[0212] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain and a light chain, wherein

[0213] (i) the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO: 32, and the light chain comprises or consists of the amino acid sequence of SEQ ID NO: 42;

[0214] (ii) the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO: 33 or 34, and the light chain comprises or consists of the amino acid sequence of SEQ ID NO: 43; or

[0215] (iii) the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO: 35 or 36, and the light chain comprises or consists of the amino acid sequence of SEQ ID NO: 44.

[0216] In one embodiment of the present invention, the amino acid changes described herein include amino acid substitutions, insertions or deletions. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.

[0217] In a preferred embodiment, the amino acid changes described herein occur in regions outside of the CDRs (e.g., in the FRs). More preferably, the amino acid changes described herein occur in regions outside of the heavy chain variable region and / or outside of the light chain variable region.

[0218] In certain embodiments, the antibodies provided herein are modified to increase or decrease the degree of glycosylation of the antibody. Addition or deletion of the glycosylation site of the antibody can be easily achieved by changing the amino acid sequence to produce or remove one or more glycosylation sites. When the antibody comprises an Fc region, the carbohydrate attached thereto can be changed. In some applications, it can be useful to remove the modification of unwanted glycosylation sites, for example, removing the fucose motif to improve antibody-dependent cellular toxicity (ADCC) function. In other applications, galactosidation can be performed to modify complement-dependent cytotoxicity (CDC).

[0219] In certain embodiments, it may be desirable to generate cysteine ​​engineered antibodies, eg, "thioMAbs," in which one or more residues of an antibody are substituted with cysteine ​​residues.

[0220] In certain embodiments, the antibodies provided herein may be further modified to contain other non-proteinaceous moieties known in the art and readily available. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0221] In some embodiments, the anti-IL1RAP antibodies or antigen-binding fragments thereof of the present invention have one or more of the following properties:

[0222] (i) exhibiting the same or similar binding affinity and / or specificity to IL1RAP as the antibodies of the present invention;

[0223] (ii) inhibiting (e.g., competitively inhibiting) the binding of an antibody of the present invention to IL1RAP;

[0224] (iii) binds to the same or overlapping epitope as an antibody of the invention;

[0225] (iv) competing with the antibodies of the present invention for binding to IL1RAP;

[0226] (v) possess one or more biological properties of an antibody of the invention.

[0227] In some embodiments, the anti-IL1RAP antibody of the present invention is an antibody in the form of IgG1, an antibody in the form of IgG2, an antibody in the form of IgG3, or an antibody in the form of IgG4, preferably, an antibody in the form of IgG1.

[0228] In some embodiments, the anti-IL1RAP antibody is a monoclonal antibody.

[0229] In some embodiments, the anti-IL1RAP antibody is humanized.

[0230] In some embodiments, the anti-IL1RAP antibody is a chimeric antibody.

[0231] In some embodiments, the anti-IL1 RAP antibodies of the invention are full-length antibodies.

[0232] In one embodiment, the anti-IL1RAP antibodies of the present invention also encompass antibody fragments thereof (e.g., antigen-binding fragments), preferably antibody fragments selected from the following: Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies such as VHH, dAb (domain antibody), bivalent antibodies or linear antibodies.

[0233] In one embodiment, the anti-IL1RAP antibodies of the present invention also encompass multispecific antibodies, such as bispecific antibodies, that specifically bind to anti-IL1RAP and one or more other antigens.

[0234] III. Nucleic acids of the present invention and host cells containing the same

[0235] In one aspect, the present invention provides nucleic acids encoding any chain, monomer, or domain of an antibody or antigen-binding fragment thereof of the present invention. Polynucleotide sequences encoding each chain can be generated using methods well known in the art. For example, when expressed from a suitable expression vector, the polypeptide encoded by the nucleic acid can exhibit human IL1RAP antigen binding ability. For example, in some embodiments, the nucleic acid encoding the variable region of the heavy chain and / or light chain is operably linked in frame with the nucleic acid encoding the constant region of the heavy chain and / or light chain, thereby producing a nucleic acid encoding the heavy chain and / or light chain of the antibody when expressed from a suitable expression vector.

[0236] In one aspect, the present invention provides a nucleic acid encoding any of the anti-IL1RAP antibodies or fragments thereof described herein. The nucleic acid may comprise a nucleic acid encoding an amino acid sequence of the light chain variable region and / or heavy chain variable region of the antibody, or a nucleic acid encoding an amino acid sequence of the light chain and / or heavy chain of the antibody.

[0237] For example, the nucleic acids of the present invention include nucleic acids encoding an amino acid sequence selected from any one of SEQ ID NOs: 13-17, 23-25, 32-36, or 42-44, or nucleic acids encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from any one of SEQ ID NOs: 13-17, 23-25, 32-36, or 42-44. As will be appreciated by those skilled in the art, due to codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by multiple nucleic acid sequences. Nucleic acid sequences encoding the molecules of the present invention can be generated using methods well known in the art, such as de novo solid-phase DNA synthesis or PCR amplification. To facilitate production and purification, a secretory signal peptide and / or a tag peptide that facilitates purification can be fused to the N-terminus of the heavy and / or light chains of the antibody.

[0238] The present invention also provides vectors comprising the nucleic acid of the present invention. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs). In preferred embodiments, the expression vector of the present invention is a pcDNA vector, such as pcDNA3.1 and / or pcDNA3.4 expression vectors.

[0239] In one embodiment, a host cell comprising the vector is provided. The present invention also provides a host cell comprising the nucleic acid or the vector. Host cells suitable for replication and support expression of the antibodies of the present invention are well known in the art. Such cells can be transfected or transduced with specific expression vectors, and large quantities of vector-containing cells can be grown for inoculating large-scale fermenters, thereby obtaining sufficient amounts of antibodies for clinical applications. Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. After expression, the antibodies can be separated from the bacterial cell paste in the soluble fraction and can be further purified.

[0240] In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells (e.g., CHO-S or CHO-K) or 293 cells (e.g., 293F or HEK293 cells)) or other cells suitable for preparing antibodies or fragments thereof. In one embodiment, the host cell is prokaryotic, for example, a bacterium, such as Escherichia coli.

[0241] For example, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors. For example, fungal and yeast strains whose glycosylation pathways have been "humanized" result in the production of antibodies with partially or fully human glycosylation patterns. Host cells suitable for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts. For example, mammalian cell lines modified to be suitable for suspension growth can be used. Other examples of useful mammalian host cell lines are monkey kidney CV1 lines (COS-7) transformed with SV40; human embryonic kidney lines (HEK293, 293F or 293T cells), etc. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells, CHO-S cells, ExpiCHO, etc.; and myeloma cell lines such as Y0, NS0 and Sp2 / 0. Mammalian host cell lines suitable for producing antibodies are known in the art.

[0242] IV. Production and Purification of Antibody Molecules of the Invention

[0243] In one embodiment, the present invention provides a method for preparing an antibody molecule or fragment thereof (preferably an antigen-binding fragment) of the present invention, wherein the method comprises culturing the host cell under conditions suitable for expressing a nucleic acid encoding an antibody molecule or fragment thereof (preferably an antigen-binding fragment) of the present invention, and optionally isolating the antibody or fragment thereof (e.g., an antigen-binding fragment). In a certain embodiment, the method further comprises recovering the antibody molecule or fragment thereof (e.g., an antigen-binding fragment) of the present invention from the host cell.

[0244] The polynucleotide encoding the polypeptide chain of the antibody of the present invention can be inserted into one or more vectors (e.g., pcDNA3.1 and / or pcDNA3.4 expression vectors) for further cloning and / or expression in a host cell. Methods well known to those skilled in the art can be used to construct expression vectors. Once an expression vector comprising one or more nucleic acid molecules of the present invention has been prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. Various techniques can be used to achieve this purpose, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection or other conventional techniques.

[0245] The antibody molecules prepared as described herein can be purified by known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, and such factors, and these will be apparent to those skilled in the art. The purity of the antibody molecules of the invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.

[0246] V. Assay

[0247] The anti-IL1 RAP antibodies or fragments thereof provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activities by various assays known in the art.

[0248] The present invention also provides assays for identifying anti-IL1RAP antibodies or fragments thereof having biological activity. Biological activity can include, for example, binding to IL1RAP (e.g., binding to human IL1RAP), inhibition of IL1RAP complex-mediated signaling pathways, prevention or treatment of inflammation, etc. Antibodies having such biological activity in vivo and / or in vitro are also provided.

[0249] For the determination of the above biological activity, please refer to the exemplary determination methods given in the Examples.

[0250] It will be appreciated that any of the above assays can be performed using the immunoconjugates of the invention in place of or in addition to an anti-IL1 RAP antibody or fragment thereof.

[0251] It will be appreciated that any of the above assays can be performed using a combination of an anti-IL1 RAP antibody or fragment thereof and an additional therapeutic agent.

[0252] VI. Immunoconjugates

[0253] In some embodiments, the present invention provides immunoconjugates comprising any of the anti-IL1 RAP antibodies provided herein and other substances, such as any active agent or label suitable for forming an immunoconjugate with the IL1 RAP antibody.

[0254] In some embodiments, the active agent suitable for forming an immunoconjugate with an IL1RAP antibody can be, for example, a chemotherapeutic agent, a toxin, a small molecule drug, a cytotoxic agent, an apoptotic agent, a chelating agent, or an immunomodulatory agent, such as an immunosuppressant.

[0255] In some embodiments, the immunoconjugate is an antibody drug conjugate, such as an ADC.

[0256] VII. Pharmaceutical Compositions and Pharmaceutical Formulations

[0257] In some embodiments, the present invention provides a composition comprising any of the anti-IL1RAP antibodies or fragments thereof (preferably antigen-binding fragments thereof) or immunoconjugates thereof described herein, preferably a pharmaceutical composition, such as a pharmaceutical formulation. In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, the composition, e.g., a pharmaceutical composition, comprises an anti-IL1RAP antibody or fragment thereof or immunoconjugate thereof of the present invention, and a combination of one or more other therapeutic agents.

[0258] The present invention also includes compositions (including pharmaceutical compositions or pharmaceutical preparations) comprising anti-IL1RAP antibodies or immunoconjugates thereof, or compositions (including pharmaceutical compositions or pharmaceutical preparations) comprising polynucleotides encoding anti-IL1RAP antibodies. In certain embodiments, the compositions comprise one or more antibodies or fragments thereof that bind to IL1RAP, or one or more polynucleotides encoding one or more anti-IL1RAP antibodies or fragments thereof. These compositions may also contain suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers.

[0259] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.

[0260] For the use of pharmaceutical excipients and their applications, see also "Handbook of Pharmaceutical Excipients", 8th edition, RC Rowe, PJ Eskey and S C Owen, Pharmaceutical Press, London, Chicago.

[0261] The compositions of the present invention can be in a variety of forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable solutions and infusible solutions), powders or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic use.

[0262] Pharmaceutical formulations comprising the antibodies described herein can be prepared by mixing the antibodies of the invention having the desired degree of purity with one or more optional pharmaceutical excipients, preferably in the form of a lyophilized formulation or an aqueous solution.

[0263] The pharmaceutical composition or preparation of the present invention can also include more than one active ingredient, the active ingredient being required for the specific indication being treated, preferably having those active ingredients of complementary activities that do not adversely affect each other. For example, it is desirable to also provide other therapeutic agents, such as cytokines, small molecule drugs, immunomodulators (such as immunosuppressants) or other antibodies, etc. The active ingredients are suitably combined in an amount effective for the intended use.

[0264] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, eg, films, or microcapsules.

[0265] VIII. Pharmaceutical Combinations and Kits

[0266] In some embodiments, the present invention also provides a pharmaceutical combination or a pharmaceutical combination product comprising an anti-IL1RAP antibody or a fragment thereof (preferably an antigen-binding fragment) of the present invention, or an immunoconjugate thereof, and one or more other therapeutic agents (e.g., cytokines, chemotherapeutic agents, small molecule drugs, immunomodulators (e.g., immune activators or immunosuppressants) or other antibodies, etc.).

[0267] Another object of the present invention is to provide a kit comprising the pharmaceutical combination of the present invention, preferably in the form of a pharmaceutical dosage unit, whereby dosage units can be provided according to a dosing regimen or a drug administration interval.

[0268] In one embodiment, the kit of parts of the present invention comprises in the same package:

[0269] - a first container containing a pharmaceutical composition comprising an anti-IL1RAP antibody or a fragment thereof;

[0270] - A second container containing a pharmaceutical composition comprising an additional therapeutic agent.

[0271] The selection of other therapeutic agents that can be combined with the molecules of the present invention depends on the use of the molecules of the present invention. For example, when the molecules of the present invention are used to treat autoimmune diseases or autoinflammatory diseases, the other therapeutic agents are any active agents suitable for treating autoimmune diseases or autoinflammatory diseases, including but not limited to cytokines, small molecule drugs, other antibodies or immunomodulators (such as immunosuppressants). For example, when the molecules of the present invention are used to treat tumors, the other therapeutic agents are any active agents suitable for treating tumors, such as anti-tumor agents or anticancer agents, including but not limited to cytokines, chemotherapeutic agents, small molecule drugs, other antibodies or immunomodulators (such as immune activators).

[0272] IX. Use and Method

[0273] In some embodiments, the method of preventing or treating a disease or condition in an individual using an antibody of the invention as a single agent or in combination comprises administering to the individual a molecule of the invention (e.g., an anti-IL1RAP antibody of the invention or an antigen-binding fragment thereof or an immunoconjugate thereof), a pharmaceutical composition, a pharmaceutical combination, or a pharmaceutical kit.

[0274] In some embodiments, the invention relates to a molecule of the invention (e.g., an anti-IL1RAP antibody or antigen-binding fragment thereof or immunoconjugate thereof), a pharmaceutical composition, a pharmaceutical combination or a kit of the invention for use in therapy, e.g., for preventing or treating a disease or condition mentioned herein.

[0275] In some embodiments, the invention relates to the use of a molecule of the invention (e.g., an anti-IL1RAP antibody or antigen-binding fragment thereof or immunoconjugate thereof), pharmaceutical composition, pharmaceutical combination or kit of the invention for the preparation of a medicament for therapy, e.g., for the prevention or treatment of a disease or condition mentioned herein.

[0276] In some embodiments, the invention relates to methods of treating a disease using a molecule of the invention (e.g., an anti-IL1RAP antibody or antigen-binding fragment thereof, or immunoconjugate thereof), a pharmaceutical composition, a pharmaceutical combination, or a kit of the invention, or uses thereof, or uses thereof for the preparation of a medicament for the treatment thereof.

[0277] In some embodiments, the disease or condition mentioned herein is a disease or condition associated with abnormal activation of a signaling pathway mediated by the IL1RAP receptor complex, or a disease or condition associated with abnormal activation of a signaling pathway mediated by IL1 (IL1α and / or IL1β), IL33 and / or IL36.

[0278] In some embodiments, the molecules of the present invention (e.g., the anti-IL1RAP antibodies or antigen-binding fragments thereof or immunoconjugates thereof of the present invention) can inhibit the activation of the downstream NF-kB signaling pathway by blocking the binding of IL1 (IL1α and / or IL1β), IL33 and / or IL36 to the IL1RAP receptor complex, thereby preventing and / or treating diseases or symptoms associated with one or more of these cytokines.

[0279] In some embodiments, patients with the diseases or conditions mentioned herein have abnormal activation of the IL1RAP receptor complex-mediated signaling pathway compared to healthy individuals.

[0280] In some embodiments, patients with the diseases or conditions mentioned herein have abnormal activation of IL1 (IL1α and / or IL1β), IL33, and / or IL36-mediated signaling pathways compared to healthy individuals.

[0281] In some embodiments, the disease or condition suitable for prevention and / or treatment according to the present invention is an autoimmune disease or an autoinflammatory disease.

[0282] In some embodiments, the diseases or conditions suitable for prevention and / or treatment according to the present invention are diseases or conditions in the rheumatic, dermatological and / or vascular fields, such as inflammation.

[0283] In some embodiments, the disease or disorder is selected from inflammation, such as peritoneal inflammation, eg, peritonitis (eg, acute peritonitis), gout (eg, acute gout), and / or skin inflammation (eg, dermatitis).

[0284] In some embodiments, the disease or condition suitable for prevention and / or treatment of the present invention is a tumor, such as cancer. In some embodiments, the tumor is a solid tumor or a hematological tumor and a metastatic lesion. In one embodiment, examples of solid tumors include malignant tumors. The cancer can be in the early, middle or late stages or be a metastatic cancer. In some embodiments, the tumor is a tumor that has escaped immune toxicity.

[0285] In some embodiments, the tumor or tumor cells have abnormally activated signaling pathways mediated by IL1 (IL1α and / or IL1β), IL33, and / or IL36, or abnormally activated IL1RAP-mediated cytokine signaling, for example, compared to healthy cells in healthy individuals or adjacent tumor cells.

[0286] In some embodiments, the tumor is an IL1RAP-positive tumor or cancer. In some embodiments, the tumor is associated with abnormal expression or abnormal activity of IL1RAP. In some embodiments, the tumor is associated with abnormal expression or abnormal activity of IL1 (IL1α and / or IL1β), IL33 and / or IL36.

[0287] In some embodiments, an IL1RAP-positive tumor or cancer refers to a subject having tumor cells expressing IL1RAP in all tumor cancers. In some embodiments, the subject's tumor cells express IL1RAP, such as moderately or highly express IL1RAP. In some embodiments, the subject has (e.g., elevated levels, such as nucleic acid or protein levels or activity) IL1RAP (e.g., compared to a healthy subject). In some embodiments, the subject has (e.g., elevated levels, such as nucleic acid or protein levels or activity) IL1RAP in a biological sample (e.g., tumor cells or tumor tissue) (e.g., compared to a biological sample of a healthy subject (e.g., a corresponding tissue or cell in a healthy subject), or compared to IL1RAP in an adjacent healthy tissue or cell of the subject).

[0288] In some embodiments, the antibodies or antibody fragments or immunoconjugates or compositions or pharmaceutical combinations or kits of the invention delay the onset of a disorder and / or symptoms associated with the disorder.

[0289] In some embodiments, the present invention provides use of an anti-IL1RAP antibody or fragment thereof, or an immunoconjugate or composition or pharmaceutical combination or kit comprising the same, in the production or preparation of a medicament for use as described herein, e.g., for preventing or treating the relevant diseases or conditions mentioned herein.

[0290] In some embodiments, the methods of prevention or treatment described herein further comprise administering to the subject or individual an antibody molecule or pharmaceutical composition or immunoconjugate disclosed herein in combination with one or more other therapies, such as therapeutic modalities and / or other therapeutic agents. In some embodiments, anti-IL1RAP antibodies or fragments thereof (as well as immunoconjugates, compositions, pharmaceutical compositions, formulations, etc. comprising the same) can also be administered in combination with one or more other therapies, such as therapeutic modalities and / or other therapeutic agents, for the purposes described herein, for example, for preventing and / or treating the relevant diseases or conditions mentioned herein.

[0291] The choice of other therapeutic agents for treatment modalities depends on the use of the molecules of the present invention. For example, when the molecules of the present invention are used to treat autoimmune diseases or autoinflammatory diseases, the other therapeutic agents are any active agents suitable for treating autoimmune diseases or autoinflammatory diseases, including but not limited to cytokines, small molecule drugs, other antibodies or immunomodulators (such as immunosuppressants or anti-inflammatory agents). For example, when the molecules of the present invention are used to treat tumors, the other therapeutic agents are any active agents suitable for treating tumors, such as anti-tumor agents or anti-cancer agents, including but not limited to cytokines, chemotherapeutic agents, small molecule drugs, other antibodies or immunomodulators (such as immune activators); the other treatment modalities are, for example, selected from surgery or radiotherapy.

[0292] In some embodiments, the antibodies described herein can be combined with other antibodies for separate administration, e.g., each as a separate antibody, or when linked (e.g., as a bispecific or multispecific antibody molecule).

[0293] The combination therapies described herein encompass combined administration (e.g., two or more therapeutic agents contained in the same formulation or separate formulations), and separate administration, in which case administration of an antibody of the invention can occur prior to, concurrently with, and / or after administration of the other therapeutic agents and / or therapies.

[0294] The antibodies or fragments thereof of the present invention (as well as immunoconjugates, compositions, pharmaceutical compositions, preparations, combination products, etc. comprising the same) can be administered by any suitable method, including parenteral administration and, if desired for local treatment, intralesional administration. Parenteral injection or infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous injection or infusion.

[0295] X. Methods and Compositions for Diagnosis and Detection

[0296] In one aspect, the present invention also relates to methods for diagnosis and detection of the antibodies or antigen-binding fragments thereof of the present invention and compositions for diagnosis and detection comprising the same.

[0297] In certain embodiments, any of the anti-IL1 RAP antibodies or fragments thereof (preferably antigen-binding fragments) provided herein can be used to detect the presence of IL1 RAP in a biological sample.

[0298] The term "detection" as used herein includes quantitative or qualitative detection, and exemplary detection methods can involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads of antibody molecule complexes, ELISA assays, PCR-techniques (e.g., RT-PCR). In certain embodiments, the biological sample is blood, serum, or other liquid samples of biological origin. In certain embodiments, the biological sample comprises cells or tissues.

[0299] In one embodiment, an anti-IL1 RAP antibody or fragment thereof is provided for use in a method of diagnosis or detection.

[0300] In another aspect, a method for detecting the presence of IL1RAP in a biological sample is provided. In certain embodiments, the method comprises detecting the presence of IL1RAP protein in the biological sample. In certain embodiments, the IL1RAP is human IL1RAP. In certain embodiments, the method comprises contacting the biological sample with an anti-IL1RAP antibody or fragment thereof as described herein under conditions that allow binding of the anti-IL1RAP antibody or fragment thereof to IL1RAP, and detecting whether a complex forms between the anti-IL1RAP antibody or fragment thereof and IL1RAP. Formation of a complex indicates the presence of IL1RAP. The method can be an in vitro or in vivo method. In one embodiment, the anti-IL1RAP antibody or fragment thereof is used to select a subject suitable for treatment with the anti-IL1RAP antibody or fragment thereof, e.g., wherein IL1RAP is a biomarker used to select the subject.

[0301] In some embodiments, a labeled anti-IL1RAP antibody or fragment thereof is provided. Labels include, but are not limited to, directly detectable labels or moieties (e.g., fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), as well as moieties that are indirectly detected, such as enzymes or ligands, e.g., via an enzymatic reaction or molecular interaction.

[0302] In some embodiments provided herein, the sample is obtained prior to treatment with an anti-IL1RAP antibody or fragment thereof. In some embodiments, the sample is obtained prior to treatment with another therapy. In some embodiments, the sample is obtained during treatment with another therapy, or after treatment with another therapy.

[0303] In some embodiments, IL1 RAP is detected prior to treatment, eg, prior to initiation of treatment or prior to a treatment after a treatment interval.

[0304] In some embodiments, a method of treating a disease of the present invention is provided, comprising: testing a subject (e.g., a sample) (e.g., a subject sample) for the presence of IL1RAP, thereby determining an IL1RAP value, comparing the IL1RAP value to a control value, and if the IL1RAP value is greater than the control value, administering to the subject a therapeutically effective amount of an anti-IL1RAP antibody or fragment thereof (e.g., an anti-IL1RAP antibody or fragment thereof described herein), optionally in combination with one or more other therapies, thereby treating the disease.

[0305] These and other aspects and embodiments of the present invention are described in the accompanying drawings (a brief description of the drawings follows) and the following detailed description of the invention and are exemplified in the following examples. Any or all of the features discussed above and throughout this application may be combined in various embodiments of the present invention. The following examples further illustrate the present invention, however, it should be understood that the examples are described in an illustrative and non-limiting manner, and that various modifications may be made by those skilled in the art. Example

[0306] Example 1. Preparation of hybridoma cells

[0307] This study utilized a hybridoma technology platform for antibody discovery. Hybridoma technology involves fusing B lymphocytes and myeloma cells to produce a cell type that retains the key characteristics of both. These two cell types are B lymphocytes from the spleen of mice immunized with an antigen, and mouse myeloma cells. The key characteristic of B lymphocytes from the spleen of mice immunized with a specific antigen is their ability to secrete antibodies, but they cannot be continuously cultured in vitro. In contrast, mouse myeloma cells can divide and proliferate indefinitely under culture conditions, a characteristic known as immortality. Under the influence of a selective culture medium, only hybrid cells formed after B lymphocytes fuse with myeloma cells can be cultured continuously, forming cell clones that possess both antibody secretion and cellular immortality.

[0308] In this experiment, the human IL1RAP extracellular domain protein was mixed with Alum or TiterMax adjuvant to immunize five groups of Balb / c mice. Four groups underwent rapid immunization (four times in 28 days) and one group underwent conventional immunization. Blood was collected after four rapid immunizations or three conventional immunizations for titer titer detection. Mice with titer titers reaching the standard were sacrificed, and spleen B lymphocytes were isolated and plated with the myeloma cell line SF (in-house acclimated). Finally, the binding of the supernatant of the fusion clone to the human IL1RAP stably transfected GS-CHO cell line was detected using FACS technology from 640 96-well fusion plates. Combining the results of functional experiments and affinity tests, 119 clones that could bind to cells and had inhibitory activity were obtained.

[0309] Table 1. Experimental animals and immunization information

[0310] Electrofusion dish preparation:

[0311] Soak the electrofusion dish thoroughly with 70% ethanol and blow dry it in a clean bench for later use.

[0312] Isolation of spleen cells: Kill the mouse by cervical dislocation, disinfect the body surface with 75% alcohol for 5 minutes, and then place it on the mouse dissection board in the clean bench, in the left lateral position, and fix the limbs with a No. 7 needle. Aseptically open the abdominal cavity to remove the spleen, wash it with basal culture medium (the configuration method is as shown in the table below), and carefully remove the surrounding connective tissue. The spleen is then transferred to another dish containing basal culture medium. Press the spleen with elbow tweezers and squeeze it with a grinding rod to fully release the spleen cells to make a spleen cell suspension. After filtering the cell suspension through a 40μM cell sieve, wash it once with 30ml of basal culture medium and centrifuge it at 1200rpm for 10 minutes.

[0313] Table 2. Preparation method of basal culture medium

[0314] Lyse red blood cells:

[0315] Remove the supernatant, resuspend the cells in 10 ml of RBC lysis buffer (Gibco), and then add 20 ml of RBC lysis buffer. Let the suspension stand for 10 minutes, then centrifuge at 1200 rpm for 10 minutes. Remove the supernatant, resuspend the cells in 10 ml of basal medium, then add 20 ml of basal medium and centrifuge at 1200 rpm for 10 minutes. Remove the supernatant, resuspend the cells in 30 ml of basal medium, and count.

[0316] Electrofusion:

[0317] Resuspend mouse myeloma SF cells (ATCC, domestically bred) in 30 ml of basal medium and count. Mix SF and spleen cells at a ratio of 1:2 to 1:1 and centrifuge at 1100 rpm for 10 minutes. Remove the supernatant and resuspend the mixed cells in 10 ml of fusion buffer (BTX press). Add 20 ml of fusion buffer, centrifuge at 1100 rpm for 10 minutes, and remove the supernatant. Resuspend the cells with an appropriate amount of fusion buffer and adjust the mixed cell density to 1×10 7 The electrofusion instrument parameters were set as follows: 9 ml of cell suspension was added to each electrofusion dish for electrofusion.

[0318] Table 3. Parameter settings of the electrofusion instrument

[0319] Plating after electrofusion:

[0320] The cells were placed in the electrofusion dish at room temperature for 15 minutes. The cells were transferred to a centrifuge tube and diluted to 1-5×10 4 B cells / ml. Add 200 μl of cell suspension to each well of a 96-well plate. Change the selection medium on day 5 or 7 after fusion. Screen after day 10 (or longer, depending on cell growth status). Identify hybridoma cells expressing specific anti-hIL1RAP antibodies using FACS (iQue screener).

[0321] Table 4. Preparation method of screening medium

[0322] Positive hybridoma cell subcloning

[0323] Subcloning step: prepare a 96-well plate, and add 200 μl of the screening medium described above to each well in rows 2 to 8. Prepare a cell suspension from the cells in the positive wells screened by the above fusion and add it to row 1. Take 100 μl of the cell suspension in row 1 and add it to row 2. Mix thoroughly and then take 100 μl and add it to the next row. Repeat the above steps until the volume of the last row becomes 300 μl; let the 96-well plate stand for 15 minutes, observe and count under a microscope. Take the volume corresponding to 100 cells and add it to 20 ml of subcloning medium AOF (STEMCELL), mix and plate, 200 μl per well. Observe under a microscope one week later, determine and mark the monoclonal wells, and pick out the positive wells to be tested.

[0324] Cell freezing: Observe the cell status. When the cells grow well and the viability is greater than 90%, centrifuge at 1000 rpm for 5 minutes and remove the supernatant. Resuspend the cells in freezing medium (90% screening medium + 10% DMSO) to 1×10 7 cells / ml, dispense into cryopreservation tubes, place in a programmed cooling box, and freeze at -80℃.

[0325] Example 2. Production and purification of chimeric antibodies

[0326] The present invention utilizes molecular biological technology to obtain the antibody sequence in anti-hIL1RAP positive hybridoma cells, and utilizes the antibody sequence to construct human-mouse chimeric antibodies.

[0327] After subcloning the 119 positive fusion cells identified in the previous steps, we further tested cell binding and functional assays in a GS-human IL1RAP stably transfected cell line, identifying 55 monoclonal antibodies. Subsequently, we sequenced these 55 clones using molecular cloning techniques. Based on sequence diversity, we obtained 33 unique sequences for constructing chimeric antibodies. Functional assays and KD affinity tests of these 33 chimeric antibodies revealed that 63C8H10 exhibited significant inhibitory effects on the IL-1, IL-33, and IL-36 pathways, resulting in the selection of 63C8H10 as the final lead molecule.

[0328] Hybridoma sequencing

[0329] RNA extraction: RNA was extracted using an RNA extraction kit (Macherey-Nagel): Carefully blow up the cells, centrifuge at 1000 rpm for 5 minutes, remove the supernatant, and the total number of cells should not exceed 5E6; add 350uL LBP, mix until clear, transfer the mixed liquid to a DNA Clearance column, centrifuge at 11,000 rpm for 30 seconds, add 100uL BS flow-through, mix; transfer the mixed liquid to an RNA column, centrifuge at 11,000 rpm for 15 seconds, and discard the liquid; add 200uL WB1, centrifuge at 11,000 rpm for 15 seconds, and discard the liquid; open the lid of the RNA column and transfer it to a new collection tube, add 600uL WB2, centrifuge at 11,000 rpm for 15 seconds, and discard the liquid; add 250uL WB2, centrifuge at 11,000 rpm for 2 minutes, discard the liquid, and centrifuge at 11,000 rpm for 1 minute; open the lid of the RNA column and transfer it to a new 1.5mL In an EP tube, evaporate ethanol for 2 minutes, add 50uL DEPC-treated ddH2O, let it stand for 2 minutes, and then centrifuge at 11,000g for 1 minute to collect the flow-through; detect RNA concentration using a Nanodrop instrument, and perform agarose gel electrophoresis to detect RNA quality.

[0330] Reverse transcription to obtain cDNA

[0331] Table 5. Preparation of reaction system I is as follows:

[0332] *From PrimeScript TM RT reagent Kit, purchased from Takara.

[0333] After slowly mixing, reverse transcription and translation were performed under the following conditions: 37°C for 15 min → 85°C for 5 min, and then cooled on ice to obtain cDNA.

[0334] The cDNA was diluted 10-fold and used as a template for the next VH\VL PCR reaction. The PCR reaction system and reaction conditions are shown in Tables 6 and 7.

[0335] Table 6. Prepare the PCR reaction system as follows:

[0336] Table 7: PCR reaction conditions are as follows:

[0337] Agarose gel electrophoresis

[0338] Prepare 1.5% agarose gel (BIOWEST), boil it, and then cool it to 50-60°C. Add nucleic acid dye at a dilution of 1:10,000, mix well, and pour it into the gel plate. Insert a comb and wait for it to solidify. Remove the comb and transfer it to the electrophoresis tank for later use.

[0339] Take 5 μL of PCR product, add 1 μL of 6× loading buffer, mix well and add to the gel wells, Marker: DL2000, and electrophoresis at voltage 160 V and current 400 mA for 20 min.

[0340] After electrophoresis, transfer the gel to the fully automatic digital gel image analysis system - Tanon, open the software, turn on the UV light source, and adjust the page zoom ratio, exposure value and focus to present a clear gel image.

[0341] The cDNA was ligated into a T vector using the Mighty TA-cloning kit (Takara) (Table 8).

[0342] Table 8: Reaction system

[0343] Transformed cells:

[0344] The competent cell culture medium (TIANGEN) was removed from -80°C and thawed on ice. 10 μL of the enzyme-linked product was added to 50 μL of the competent cell culture medium and incubated on ice for 30 min. The cells were heat-shocked at 42°C for 90 s, and the tubes were quickly transferred to ice and allowed to stand for 3 min. 1 mL of resistance-free LB medium (Sangon Biotech (Shanghai) Co., Ltd.) was added to the tubes and incubated on a shaker at 220 rpm / min at 37°C for 60 min. The tubes were then coated with LBA plates and incubated overnight at 37°C. Clones were then selected for sequencing.

[0345] Construction of chimeric antibodies

[0346] The VH and VL regions of the mouse anti-hIL1RAP antibody produced by the hybridoma in Example 1 that had been sequenced were amplified by PCR.

[0347] The PCR amplification products were recovered by gel excision.

[0348] Homologous recombination reaction:

[0349] The homologous recombination system is as follows (Table 9).

[0350] Table 9: Homologous recombination system

[0351] React at 37°C for 40 minutes to obtain the recombinant product. Remove the DH5α competent medium from -80°C and thaw on ice. Add 50 μL of competent medium to 10 μL of the enzyme-linked product and incubate on ice for 30 minutes. Heat shock the tube at 42°C for 120 seconds, quickly transfer the tube to ice, and let it rest for 3 minutes. Add 1 mL of LB medium and incubate on a shaker at 37°C for 1 hour. Centrifuge at 8000 rpm / min for 30 seconds, discard the supernatant, and reserve 100 μL of medium. Pipet and mix the cells thoroughly, then spread them onto LBA plates. Single clones are selected for sequencing. Positive clones are selected as those containing the plasmid inserted in the correct orientation and are saved.

[0352] Expression and purification of chimeric antibodies

[0353] A plasmid containing the anti-hIL1RAP antibody was extracted from the positive clones obtained above. The plasmid was transfected into Expi293F cells, and the target protein was purified by the following steps:

[0354] Expi293F cells (purchased from Gibco) were cultured with Expi293F medium (Gibco, REF#A14351-01). The cell density was checked one day before transfection (viability should be greater than 95%) and adjusted to 3 × 10 6 The cell density was adjusted to 3 × 10 cells / ml on the day of transfection. 6 cells / ml.

[0355] Take 1 / 10 of the final transfection volume of Opti-MEM medium (Gibco, REF#31985-070) as the transfection buffer, add the DNA to be transfected at a ratio of 1 mg / L, where the light and heavy chain plasmids are in a 1:1 ratio, mix well, add PEIMax (Polysciences Inc. Cat#24765-1) at a DNA:PEI mass ratio of 1:3, mix well, incubate at room temperature for 20 minutes, and then gently pour the mixture into the Expi293F cell suspension while shaking. The cells are cultured in a shaker under the conditions of 8% CO2, 36.5°C, and 120 rpm.

[0356] After 16-18 hours of culture, the cell suspension was supplemented with 2% (v / v) of 200 g / L feed (100 g / L Phytone Peptone + 100 g / L Difco Select Phytone), a glucose solution to a final concentration of 5 g / L, and valproic acid sodium salt (Merk, Cat# P4543-100G) to a final concentration of 2.2 mM. The suspension was gently mixed and cultured for another 7 days at 8% CO₂, 36.5°C, and 120 rpm. Samples were then collected after centrifugation and filtration using a 0.22 μm disposable vacuum filter. The antibody was affinity captured using a HiTrap MabSelect PrismA (GE Healthcare, Cat#17549853) affinity chromatography column. Before purification, 10-20 column volumes of 0.1 M NaOH were passed through the tubing and affinity chromatography column, and then the tubing and column were washed with 10-20 column volumes of distilled water. The packed column was equilibrated with 5 column volumes of 1× PBS (Gibco). The filtered cell feed was passed through the column, and the packed column was washed with 10 column volumes of 1× PBS to remove nonspecifically bound proteins. The packed column was rinsed with 5 column volumes of elution buffer (100 mM sodium citrate, pH 3.5), and the eluate was collected, adjusted to pH 6.0 with 2 M Tris, filtered and sterilized, and submitted for SEC analysis.

[0357] The CDRs, light chain variable regions, heavy chain variable regions, amino acid sequences of the light chain and heavy chain of a chimeric antibody obtained in the present invention, as well as the sequence numbers are shown in Table ABCD.

[0358] The control drugs used in the present invention are the IL1 receptor antagonist Anakinra (IL1Ra), and the control antibodies anti-human IL1RAPCAN10, REGN3500 and BI655130. Their molecular sequences, antibody CDRs, light chain variable regions and heavy chain variable regions, and amino acid sequences of the light and heavy chains are also shown in Tables BCDE.

[0359] Similar steps as above were used to generate anti-CAN10 antibody (synthesized in-house), REGN3500 antibody (synthesized in-house), and BI655130 antibody (Bio-Ying Bio).

[0360] Example 3. Determination of the binding kinetics between the chimeric antibody of the present invention and the antigen using biointerferometry

[0361] The equilibrium dissociation constants (KD) of the antibodies of the present invention for binding to human, cynomolgus monkey, and mouse IL1RAP were determined using biointerferometry (ForteBio). ForteBio affinity determinations were performed according to existing methods (Estep, P et al., High throughput solution-based measurement of antibody antigen affinity and epitope binning. MAbs, 2013. 5(2): p. 270-8).

[0362] Half an hour before the start of the experiment, according to the number of samples, an appropriate number of AHC (18-5060, Sartorius) sensors were taken and immersed in SD buffer (PBS 1×, BSA 0.1%, Tween 20 0.05%).

[0363] 100 μl of SD buffer, antibody, and antigen (human IL1RAP (ILP-H5225, Acro Biosystems), mouse IL1RAP (Sino Biological, 52657-M08H), and cynomolgus macaque IL1RAP (Sino Biological, 90934-C08H)) were added to a 96-well black polystyrene half-well microplate (Greiner, 675076). The plate was arranged according to the sample position and the sensor position was selected. Instrument parameters were set as follows: Run steps: Baseline, Loading ~1 nm, Baseline, Association, and Dissociation; the run time for each step depended on the sample association and dissociation rates. The rotation speed was 1000 rpm and the temperature was 30°C. KD values ​​were analyzed using ForteBio analysis software.

[0364] In the experiments described in the above assays, the affinities of antibodies 63C8H10 and CAN10 are shown in Table 10:

[0365] Table 10. Affinity constants (equilibrium dissociation constants) of antigen-antibody binding assays by ForteBio

[0366] In the above experiments, the KD values ​​of the chimeric antibody 63C8H10 against humans and cynomolgus macaques were 4.84E-10M and 6.47E-10M, respectively. Compared with the control antibody CAN10, the antibodies in this study had similar or better KD values.

[0367] Example 4. Humanization of chimeric antibodies

[0368] The chimeric antibody obtained in Example 1 was humanized. The antibody humanization process was performed using the Discovery Studio proprietary software program from Dassault Systèmes. First, the sequence was entered into the software, which generated a three-dimensional model of the sequence. Humanization was then performed through the following steps:

[0369] ① Determine the CDR loop structure;

[0370] ② Find the closest homologous sequence for each V / J region of the heavy and light chains in the human germline sequence database;

[0371] ③ Screening for the human germline that best matches the heavy and light chains and the lowest amount of back mutations;

[0372] ④ Constructing the CDR region of the chimeric antibody onto the human framework region;

[0373] ⑤ Using sequence and structural features, determine the amino acid positions in the framework region that maintain CDR function;

[0374] ⑥ Perform back mutation at the sequence position determined to be important (return to the input amino acid type);

[0375] ⑦Generate a three-dimensional model of the humanized sequence;

[0376] ⑧Manually inspect the sequence and structure to identify risk sites that may cause misfolding or reduced stability;

[0377] ⑨Optimize amino acids at risk sites.

[0378] The CDRs, light chain variable regions, heavy chain variable regions, and amino acid sequences of the light and heavy chains of the four humanized antibodies (HZ63C8.4, HZ63C8.5, HZ63C8.6, and HZ63C8.7) obtained in the present invention are shown in Tables AE below.

[0379] Example 5. ForteBio determination of binding kinetics between humanized antibodies and antigens

[0380] The equilibrium dissociation constants (KD) for binding of humanized antibodies of different Fc subtypes of the present invention to human IL1RAP were determined using the ForteBio affinity assay. The ForteBio affinity assay method was the same as in Example 2. In the experiments described in the above assay, the affinities of antibodies hz63C8.4, hz63C8.5, hz63C8.6, and hz63C8.7 are shown in Table 11:

[0381] Table 11. Affinity constants for antigen-antibody binding assays performed by ForteBio.

[0382] In the above experiments, the KD values ​​of the humanized antibodies hz63C8.4, hz63C8.5, hz63C8.6, and hz63C8.7 described in this article were 4.54E-10M, 5.10E-10M, 3.99E-10M, and 4.12E-10M, respectively. Compared with the control group CAN10, the humanized antibodies in this study had similar or better KD values.

[0383] Example 6. Binding experiment of antibodies to cells overexpressing IL1RAP

[0384] In this study, flow cytometry was used to detect the binding of the chimeric antibody and humanized antibody of the present invention at gradient dilution to a CHO stable cell line overexpressing human IL1RAP on its surface (CHO-hIL1RAP).

[0385] A CHO cell line overexpressing human IL1RAP (CHO-hIL1RAP) was established by transfecting a full-length human IL1RAP plasmid (GenWeiZhi) into CHO-GS cells (Lonza).

[0386] CHO-hIL1RAP cells were counted and diluted to 1×10^6 cells / ml. 100 μl / well of the culture medium was added to a U-bottom 96-well plate. After centrifugation at 400g for 5 minutes, the cell culture medium was removed. Chimeric antibody ch63C8 and humanized antibodies hz63C8.4, hz63C8.5, and hz63C8.6 (8 concentrations tested: highest antibody concentration was 100 nM in two-fold dilutions or 300 nM in three-fold dilutions in PBS containing 0.1% bovine serum albumin (BSA)) were added to the plate and the cells were resuspended in 100 μl / well. The cells were incubated on ice for 30 minutes. After centrifugation at 400g for 5 minutes, the supernatant was removed and the cells were washed once with PBS. The PBS was removed after centrifugation at 400g for 5 minutes, and 100 μl of a 1:200 diluted PE-conjugated secondary antibody (Biolegend, Cat. No. 410708) was added to each well. Incubate on ice in the dark for 30 minutes. Centrifuge at 400g for 5 minutes, remove the supernatant, and wash the cells once with PBS. Resuspend the cells in 100 μl of 1× PBS and analyze by FACS. The binding of the chimeric antibody ch63C8 and the humanized antibodies hz63C8.4, hz63C8.5, and hz63C8.6 to CHO-hIL1RAP cells is shown in Figure 1.

[0387] In the above experiments, the chimeric antibody ch63C8 and the humanized antibodies hz63C8.4, hz63C8.5, and hz63C8.6 were all able to bind to human IL1RAP overexpressed in CHO cells, with EC50 values ​​of 6.96 nM, 5.94 nM, 9.79 nM, and 4.64 nM, respectively.

[0388] Example 7. Detection of Antibody Biological Activity in Fluorescent Reporter Cell Lines

[0389] Anti-IL1RAP antibodies can inhibit the activation of the downstream NF-kB signaling pathway by blocking the binding of IL1α, IL1β, IL33, and IL36 to the IL1RAP receptor complex. This study used three fluorescent reporter cell lines provided by Nearshore Protein: IL1 pathway activity reporter cells (293-IL1 Res cell line, XCC10), IL33 pathway activity reporter cells (293-IL33 Res cell line, XCC16), and IL36 pathway activity reporter cells (293-IL36 Res cell line, XCC15-1). According to the method provided in the instructions, the expression of fluorescent reporter genes was detected to reflect the activation of NF-kB signaling, thereby detecting the inhibitory effect of antibody binding to IL1RAP.

[0390] Detecting the biological activity of antibodies against the IL1 pathway in fluorescent reporter cell lines

[0391] Cell recovery: 1) Gently shake in a 37°C water bath to thaw frozen cells, keeping the seal and bottle cap above the water surface to reduce the risk of contamination. Thawing should be done as quickly as possible (within 1 minute); 2) Immediately after thawing, disinfect the cells with 75% alcohol and move them to a clean bench. Strict aseptic operation should be followed; 3) Move the cell suspension to a 10ml preheated subculture medium (FreeStyle TM 293 Expression Medium) in a centrifuge tube, resuscitate and passage three times, then add Neomycin (100 μg / ml); 4), centrifuge at 800-1000 rpm (RCF 200-300g) for 5 min; 5), discard the supernatant and resuspend the cells in 1 ml of passage medium; 6), transfer the cells to a 125 ml shake flask containing 19 ml of passage medium; 7), culture in a shaker at 37°C, 7% CO2, and 120 rpm.

[0392] Cell culture: Maintain Neomycin pressure (100ug / ml) during subculturing; subculture every 1-2 days; the subculture density should be controlled at 0.3-0.5×10^6 cells / ml.

[0393] Activity detection:

[0394] 1) Cell plating: Count the cells and adjust the cell density to 3×10^5 cells / ml. Add 100 μl of cells to a 96-well plate and culture in a 37°C incubator overnight.

[0395] 2) Antibody and cytokine processing: using FreeStyle TMDilute the hz63C8.6 antibody, HEL (negative control), and CAN10 test samples to 1.2 μM (final concentration 300 nM) in 293 medium. Then, dilute each sample three-fold to a total of 11 concentrations. Add 50 μL of each diluted sample to a 96-well plate containing cells and incubate in a 37°C incubator for 1 hour. Dilute human IL-1B (Acro Biosystems, ILB-H4110-50 μg) to 4 ng / mL (final concentration 1 ng / mL) and add 50 μL to each well for a final volume of 200 μL. Incubate for 6 hours.

[0396] 3) Fluorescence Detection: Mix the Bio-Lite Luciferase Assay System (Nanjing Novizan, Cat. No. DD1201-02) lysis buffer with the substrate and equilibrate at room temperature for 30 minutes. Centrifuge the 96-well plate at 300g for 5 minutes. Gently aspirate 100µl of the supernatant and add 100µl of the Bio-Lite Luciferase Assay System 1X lysis buffer (1:1 volume ratio). Gently shake the remaining 100µl plate and let it stand for at least 10 minutes before detecting with the instrument.

[0397] In the above experiment, the experimental results are shown in Figure 2. Antibody hz63C8.6 can effectively block the activation of IL1β-mediated downstream signaling pathways, and the inhibitory effect is comparable to that of benchmark CAN10 (Cantargia).

[0398] Detecting the biological activity of antibodies against the IL36 pathway in fluorescent reporter cell lines

[0399] Cell Culture: The IL36 pathway fluorescent reporter cell line 293-IL36Res (Nearshore Protein, XCC15-1) was cultured in DMEM medium supplemented with 10% FBS. No antibiotics were added for the first three passages after recovery. Cells were incubated at 37°C in a 5% CO2 incubator. After three passages, antibiotics (25 μg / ml Hygro + 400 μg / ml G418) were added and continued to culture. Generally, after 2-3 additional passages, cells were used for experiments. After digestion with TrypLE (GIBCO, 12605028), the cells were counted and the cell density was adjusted to 3E5 / ml with culture medium (90% DMEM + 10% FBS + 25 μg / ml Hygro + 400 μg / ml G418). 100 μl of the cell suspension was added to each well of a 96-well luminescent plate with a white bottom and a transparent cover (Shanghai Wohong, WHB-96-03) and cultured overnight. The next day, each test sample was diluted to 1.2 μM (final concentration 300 nM) in culture medium and then diluted three-fold for a total of 11 concentrations. According to the experimental design, 50 μl of each of the corresponding concentrations of benchmark CAN10 and anti-IL1RAP antibody hz63C8.6 were added and incubated in an incubator for 1 hour. Next, IL36a (Novoprotein, CR61) was diluted to 400 ng / ml (final concentration 100 ng / ml) in culture medium and 50 μl was added to each well for a final volume of 200 μl. The cells were incubated in an incubator for 6 hours. Centrifuge at 300 g for 5 minutes, discard 100 μl of supernatant, add 100 μl of the solution, and lyse the cells using a Bio-Lite Luciferase Assay system for 5-10 minutes before analysis.

[0400] In the above experiment, the experimental results are shown in Figure 3. Antibody hz63C8.6 can effectively block the activation of IL36-mediated downstream signaling pathways, and the inhibitory effect is comparable to that of benchmark CAN10 (Cantargia).

[0401] Detecting the biological activity of antibodies against the IL33 pathway in fluorescent reporter cell lines

[0402] Cell Culture: IL33 pathway fluorescent reporter cell line 293-IL33 Res (XCC16) was cultured in DMEM medium supplemented with 10% FBS. No antibiotics were added for the first three passages after recovery. Cells were incubated at 37°C in a 5% CO2 incubator. After three passages, antibiotics (6.25 μg / ml Hygro + 12.5 μg / ml G418) were added and continued to culture. Generally, after 2-3 additional passages, cells were used for experiments. After digestion with TrypLE (GIBCO, 12605028), the cells were counted and the cell density was adjusted to 3E5 / ml with culture medium (90% DMEM + 10% FBS + 6.25 μg / ml Hygro + 12.5 μg / ml G418). 100 μl of the cell suspension was added to each well of a 96-well luminescent plate with a white bottom and a transparent cover (Shanghai Wohong, WHB-96-03) and incubated overnight. The next day, each test sample was diluted to 1.2 μM (final concentration 300 nM) in culture medium and then diluted three-fold for a total of 11 concentrations. 50 μl of each of the corresponding concentrations of benchmark CAN10 and anti-IL1RAP antibody hz63C8.6 were added to the wells and incubated in an incubator for 1 hour. Next, human IL33 (PeproTech, 200-33-100UG) was diluted to 80 ng / ml (final concentration 20 ng / ml) in culture medium and 50 μl was added to each well for a final volume of 200 μl. The wells were incubated in an incubator for 6 hours. After centrifugation at 300 g for 5 minutes, 100 μl of supernatant was discarded and lysed with 100 μl of the Bio-Lite Luciferase Assay system for 5-10 minutes before analysis.

[0403] In the above experiment, the experimental results are shown in Figure 4. Antibody hz63C8.6 can effectively block the activation of IL33-mediated downstream signaling pathways, and the inhibitory effect is better than the benchmark CAN10 (Cantargia).

[0404] Example 8. Verification of Antibody Activity on IL36 Signaling Pathway in Squamous Cell Carcinoma Cell Line A431

[0405] Cell Culture: A431 squamous cell carcinoma cells (ATCC, CRL-1555) were cultured in DMEM medium supplemented with 10% FBS. Cells were incubated at 37°C in a 5% CO2 incubator. After 2-3 additional passages, cells were counted and adjusted to a cell density of 1E5 / ml using culture medium (90% DMEM + 10% FBS + 1% Pen / Strep). 100 μl of the cell suspension was added to each well of a 96-well luminescent plate with a white bottom and a transparent cover (Shanghai Wohong, WHB-96-03) and incubated overnight. The next day, each test sample was diluted to 400 nM (final concentration 100 nM) using culture medium, followed by subsequent 10-fold dilutions for a total of 6-7 concentrations. According to the experimental design, 50 μl of each of the corresponding concentrations of benchmark CAN10 (Cantargia), BI655130, and humanized antibodies hz63C8.4, hz63C8.5, hz63C8.6, and hz63C8.7 were added and pretreated in an incubator for 1 hour. Then, 50 μl of the cytokine human IL-36a (Novoprotein, CR61) was diluted to 400 ng / ml (final concentration 100 ng / ml) in culture medium and added to each well for a final volume of 200 μl. The cells were incubated in an incubator for an additional 24 hours. After centrifugation at 300 g for 5 minutes, the supernatant was collected. The supernatant was diluted threefold and the IL-8 concentration in the cell supernatant was determined according to the instructions of the Human IL-8 ELISA Ready-SET-Go Kit (eBioscience, 88-8086-88).

[0406] In the above experiments, as shown in FIG5 , the experimental results show that both the chimeric antibody and the humanized antibody can effectively block the activation of the downstream signaling pathway mediated by IL36 in epithelial cells.

[0407] Example 9. Detection of Antibody Inhibitory Activity on IL-1 Pathway in PBMC Cells

[0408] PBMCs were rapidly thawed in a 37°C water bath and added to RPMI 1640 complete medium (10% FBS, 1% Pen Strep, 10% Sodium Pyruvate, 1% 2-Me) containing DNase I. Centrifuge at 250 g for 7 min, discard the supernatant, add the above complete medium for washing, and centrifuge again. Repeat this process twice. Resuspend the cells in RPMI 1640 complete medium and adjust the cell density to 2 x 10^6 cells / ml. Plate 100 μl per well in a 96-well round-bottom plate (Corning, 3799). Benchmark protein anti-IL1RAP antibody CAN10 (Cantargia), Anakinra (IL1R antagonist, IL1Ra, Medchemexpress, HY-108841), anti-IL1RAP chimeric antibody 63C8H10, and humanized antibodies hz63C8.4, hz63C8.5, hz63C8.6, and hz63C8.7 were diluted to various concentrations. Each antibody was diluted to 4000 nM (final concentration 1000 nM) and then diluted 3-fold for 11 points, or 400 nM (final concentration 100 nM) was diluted 10-fold for 6 points. 50 μl of each was added to the corresponding wells and pretreated for 1 hour. Next, human IL-1β (Acro Biosystems, ILB-H4110-50 μg) and IL-1α (Novoprotein, C070) were diluted to 400 ng / ml (final concentration 100 ng / ml) and 50 μl of each diluted IL-1β and IL-1α were added to corresponding 96-well plates. The final volume per well was 200 μl. After 24 hours of co-stimulation, the supernatant was collected and TNFα secretion was assayed by ELISA.

[0409] The experimental results are shown in Figure 6. Both the chimeric antibody and the humanized antibody can potently inhibit the IL-1 signaling pathway in PBMC cells, and the inhibitory effect is stronger than that of the marketed IL-1 pathway drug Anakinra and the antiIL1RAP benchmark CAN10.

[0410] Example 10. Detection of Antibody Inhibitory Activity on IL-36 Pathway in PBMC Cells

[0411] PBMCs were rapidly thawed in a 37°C water bath and added to RPMI 1640 complete medium (10% FBS, 1% Pen Strep, 10% Sodium Pyruvate, 1% 2-Me) containing DNase I. Centrifuge at 250 g for 7 min, discard the supernatant, add the above complete medium for washing, and centrifuge again. Repeat this process twice. Resuspend the cells in RPMI 1640 complete medium and adjust the cell density to 2 x 10^6 cells / ml. Plate 100 μl per well in a 96-well round-bottom plate (Corning, 3799).

[0412] Benchmark anti-IL1RAP antibodies CAN10 (Cantargia), BI 655130 (anti-IL36R), and humanized anti-IL1RAP antibodies hz63C8.4, hz63C8.5, hz63C8.6, and hz63C8.7 were diluted to various concentrations. Each antibody was diluted to 400 nM (final concentration 100 nM) and then serially diluted 10-fold to six different concentrations. 50 μl of each was added to the corresponding wells for 1 hour of pretreatment. Next, human IL36α (Novoprotein, CR61) was diluted to 400 ng / ml (final concentration 100 ng / ml) and 50 μl of each diluted IL36α was added to the corresponding 96-well plates. The final volume per well was 200 μl. After 24 hours of co-stimulation, the supernatant was collected and TNFα secretion was assayed by ELISA.

[0413] The above experimental results are shown in Figure 7. The humanized anti-IL1RAP antibody can effectively inhibit the IL-36 signaling pathway in PBMC cells, and the inhibitory effect is stronger than the anti-IL36R benchmark BI 655130.

[0414] Example 11. Detection of Antibody Inhibitory Activity on IL-33 Pathway in PBMC Cells

[0415] PBMCs were rapidly thawed in a 37°C water bath and added to RPMI 1640 complete medium (10% FBS, 1% Pen Strep, 10% Sodium Pyruvate, 1% 2-Me) containing DNase I. Centrifuge at 250 g for 7 min, discard the supernatant, add the above complete medium for washing, and centrifuge again. Repeat this process twice. Resuspend the cells in RPMI 1640 complete medium and adjust the cell density to 2 x 10^6 cells / ml. Plate 100 μl per well in a 96-well round-bottom plate (Corning, 3799).

[0416] Benchmark proteins, including the anti-IL1RAP antibody CAN10 (Cantargia), REGN3500 (anti-IL33), the chimeric anti-IL1RAP antibody 63C8H10, and the humanized antibodies hz63C8.4, hz63C8.5, hz63C8.6, and hz63C8.7, were diluted to various concentrations. Each antibody was diluted to 400 nM (final concentration 100 nM) and then 10-fold diluted to 6-7 different concentrations. 50 μl of each was added to the corresponding wells for 1 hour of pretreatment. Then, the cytokines IL33 (PeproTech, 200-33-100UG) were diluted to 80 ng / ml (final concentration 20 ng / ml) and IL12 (Nearshore Protein, GMP-CI58) were diluted to 8 ng / ml (final concentration 2 ng / ml). 50 μl of each diluted IL33+IL12 mixture was added to the corresponding 96-well plate. The final volume per well was 200 μl. After 24 hours of co-stimulation, the supernatant was collected and the secretion of IFNγ was detected by ELISA.

[0417] The experimental results are shown in Figure 8. Both the chimeric antibody and the humanized antibody can inhibit the IL33 signaling pathway in PBMC cells, and the inhibitory effect is stronger than that of the anti-IL1RAP antibody benchmark CAN10, and weaker than that of the anti-IL33 antibody REGN3500.

[0418] Example 12. Pharmacokinetic characteristics of subcutaneous administration in mice

[0419] Balb / c mice were subcutaneously injected with the anti-IL1RAP antibody hz63C8.6 (10 mg / kg). Peripheral blood was collected at 0, 0.5, 2, 6, 24, 48, 96, 192, 336, 504, 672, 840, and 1008 hours after administration. After clotting at room temperature for at least 3 hours, serum was collected by centrifugation (room temperature, 5000 rpm, 5 minutes). Anti-IL1RAP levels in serum at different time points were measured by ELISA.

[0420] Results showed that anti-IL1RAP exhibited favorable pharmacokinetic characteristics in mice (Table 12). Even after 42 days of monitoring, plasma concentrations did not drop below 1 / 10 of Cmax. Based on the data obtained, T1 / 2 was calculated to be 316 hours, and clearance (CL) was 0.23 ml / kg / h (Table 12).

[0421] Table 12: Pharmacokinetic study parameters of anti-IL1RAP antibody (hz63C8.6) administered subcutaneously to mice

[0422] Example 13. Pharmacokinetic characteristics of intravenous administration in mice

[0423] In Balb / c mice, the anti-IL1RAP antibody hz63C8.6 molecule was administered by tail vein injection (10 mg / kg), and peripheral blood was collected at 0, 0.083, 0.5, 2, 6, 24, 48, 96, 168, 336, and 504 hours after administration, and serum was collected. The anti-IL1RAP content in the peripheral blood was detected by ELISA.

[0424] The results showed that anti-IL1RAP exhibited favorable pharmacokinetic characteristics in mice (Table 13). The monitoring period lasted 504 hours, and based on the data obtained, the T1 / 2 was calculated to be 192 hours and the clearance rate (CL) was 0.45 ml / kg / h (Table 13).

[0425] Table 13: Pharmacokinetic study parameters of anti-IL1RAP antibody (hz63C8.6) administered intravenously to mice

[0426] Example 14. Analysis of antibody physicochemical properties

[0427] In this study, IBI303 (adalimumab) and IBI310 (ipilumab) were used as control antibodies. IBI303 has better drugability, while IBI310 has poorer drugability.

[0428] 1) SMAC column detection of antibody colloidal stability

[0429] The antibody colloidal stability was tested by recording the retention time of the antibody in the Zenix-HPLC column.

[0430] 2) HIC column detection of antibody colloid hydrophobicity

[0431] The hydrophobicity of the antibody was detected by recording the retention time of the antibody in the HIC-HPLC column.

[0432] 3) CIC detection antibodies are non-specific

[0433] IgG from human serum was coated onto an NHS-activated column, and the retention time of the antibody on the column was examined by HPLC. Retention time showed a significant negative correlation with solubility, thus enabling the screening of antibodies with better solubility.

[0434] 4) DLS detection of thermal stability of antibodies

[0435] Dynamic light scattering (DLS) uses laser light to illuminate small particles undergoing Brownian motion in a solution and detects changes in the intensity of the scattered light. DLS can be used to determine the particle size of proteins in solution and their stability under varying temperatures or concentrations.

[0436] 5) DSC detection of thermal stability of antibodies

[0437] Differential Scanning Calorimetry (DSC) is a thermal analysis method that measures the energy difference between a sample and a reference substance as it changes with temperature under programmed temperature conditions. As the temperature rises, the protein structure changes, and the accompanying heat changes are recorded by the differential scanning calorimeter as a DSC curve. The Tm value of the protein is determined by analyzing the DSC curve.

[0438] Table 14. Antibody thermal stability test results

[0439] Example 15. Verification of the biological activity of antibodies in a mouse acute peritonitis model

[0440] Male IL1RAP humanized mice (B-hIL1RACP mice, Biomice, Cat. No: 110072) with a C57BL / 6 background, 6-8 weeks old, were randomly divided into four groups after 1 week of adaptive feeding: untreated group, Anakinra treatment group, hz63C8.6 treatment group and PBS treatment group.

[0441] Among them, the last three groups were respectively injected with Anakinra (10mg / kg, Medchemexpress, HY-108841), hz63C8.6 (20mg / kg) and PBS (200ul) through the tail vein. One hour later, monosodium urate crystals (MSU, InvivoGen, tlrl-msu-25) were injected intraperitoneally at 3mg / mouse to induce an acute peritonitis model. Six hours later, peritoneal cells were flushed with cold PBS, with 10ml per mouse. The collected liquid was placed in a 15ml centrifuge tube. After all mice were flushed, the tube was centrifuged at 300g, 4℃, and centrifuged for 5 minutes. The supernatant was discarded and the cell pellet was resuspended in PBS containing 0.5% BSA. After cell counting, flow cytometry staining was performed to detect changes in various immune cells.

[0442] The results showed that hz63C8.6 treatment significantly alleviated peritoneal inflammation in mice, significantly reduced the recruitment of inflammatory cells in the peritoneal cavity (Figure 9), and significantly reduced the number of neutrophils (Figure 9). Furthermore, hz63C8.6 showed a more potent inhibitory effect than Anakinra.

[0443] Example 16. Verification of the biological activity of antibodies in a mouse gout model

[0444] Male IL1RAP humanized mice (B-hIL1RACP mice, Biomice, Cat. No: 110072) on a C57BL / 6 background, aged 8-10 weeks, were acclimated for 1 week and then randomly divided into four groups: untreated, Anakinra-treated, hz63C8.6-treated, and PBS-treated. The latter three groups were injected with Anakinra (10 mg / kg, Medchemexpress, HY-108841), hz63C8.6 (20 mg / kg), and PBS (200 μl) via the tail vein. One hour later, monosodium urate crystals (MSU, InvivoGen, tlrl-msu-25) were injected into the paw at 1.8 mg / 50 μl per mouse to induce an acute gout model.

[0445] After 24 hours, the redness and swelling of the mice's paws were observed, and the thickness of the paws was measured. The results showed that the hz63C8.6 treatment group significantly alleviated gout and inhibited the thickening of the mice's paws, and the inhibitory effect was superior to Anakinra (Figure 10).

[0446] Example 17. Verification of the biological activity of the antibody in a mouse dermatitis model

[0447] Male IL1RAP and IL36R double humanized mice (B-hIL36R / hIL1RACP mice, Biomice, Cat.No: 112383) on a C57BL / 6 background, 6-8 weeks old, were adaptively reared for 1 week and randomly divided into five groups: untreated group, anti-IL36R treatment group (BI 655130 group), anti-IL1RAP antibody bench mark control group (CAN10 group), hz63C8.6 treatment group, and PBS treatment group.

[0448] The last four groups received tail vein injections of BI655130 (10 mg / kg), CAN10 (10 mg / kg), hz63C8.6 (10 mg / kg), and PBS (200 μl). Then, on days 1, 3, 5, and 7 after administration, the ear skin was injected with human IL-36γ cytokine (Novoprotein, CM77-500g) at a rate of 2 μg / 25 μl per mouse to induce an ear dermatitis model. Changes in ear thickness were measured at each injection.

[0449] The results showed that hz63C8.6 significantly inhibited human IL36γ-induced mouse skin inflammation, as evidenced by a reduction in mouse skin thickness. Its inhibitory effect was comparable to that of BI 655130 and significantly stronger than that of CAN10 (Figure 11).

Claims

1. An anti-IL1RAP antibody or an antigen-binding fragment thereof, comprising: The three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in any one of SEQ ID NOs: 13-17, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in any one of SEQ ID NOs: 23-25.

2. An anti-IL1RAP antibody or an antigen-binding fragment thereof, comprising a first heavy chain complementary determining region (HCDR1), a second heavy chain complementary determining region (HCDR2), a third heavy chain complementary determining region (HCDR3), and a first light chain complementary determining region (LCDR1), a second light chain complementary determining region (LCDR2), and a third light chain complementary determining region (LCDR3), wherein the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, and LCDR3 respectively comprise or consist of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO:

6.

3. The anti-IL1RAP antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH), wherein the heavy chain variable region comprises, consists of, or comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 13-17, or consists of said sequence; and / or the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL), wherein the light chain variable region comprises, consists of, or comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 23-25, or consists of said sequence. The amino acid sequence shown in any one of NO:23-25, or consisting of said sequence.

4. An anti-IL1RAP antibody or an antigen-binding fragment thereof, comprising (i) a VH comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 16 or 17, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and a VL comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 25, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; (ii) a VH comprising, or consisting of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and a VL comprising, or consisting of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, as shown in SEQ ID NO: 23; or (iii) a VH comprising, or consisting of, an amino acid sequence represented by SEQ ID NO: 14 or 15, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and a VL comprising, or consisting of, an amino acid sequence represented by SEQ ID NO: 24, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

5. The anti-IL1RAP antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising a heavy chain variable region and a light chain variable region, wherein (i) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16 or 17, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 25; (ii) the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 16 or 17, and the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 25; (iii) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 13, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 23; (iv) the heavy chain variable region consists of the amino acid sequence set forth in SEQ ID NO: 13, and the light chain variable region consists of the amino acid sequence set forth in SEQ ID NO: 23; (v) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 14 or 15, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 24; or (vi) the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 14 or 15, and the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:

24.

6. The anti-IL1RAP antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, comprising an Fc region, for example, the Fc region is derived from an Fc region of IgG1, IgG2, IgG3 or IgG4, for example, an Fc region of human IgG1, IgG2, IgG3 or IgG4; and / or The antibody or antigen-binding fragment thereof comprises a heavy chain constant region, and the heavy chain constant region is derived from the constant region of IgG1, IgG2, IgG3 or IgG4, such as the constant region of human IgG1, IgG2, IgG3 or IgG4, such as the IgG1 heavy chain constant region (i) comprises or consists of the amino acid sequence of SEQ ID NO: 50; or (ii) comprising an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 50; Optionally, the heavy chain constant region or Fc region lacks a C-terminal lysine; and / or The heavy chain constant region or Fc region comprises a mutation that reduces binding to Fcγ receptors, such as L234A / L235A mutation and / or P329G mutation, such as L234A / L235A mutation or L234A / L235A mutation and P329G mutation; for example, the heavy chain constant region (i) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 51 or 52; (ii) comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 51 and has an L234A / L235A mutation; or (iii) comprising an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 52, and having an L234A / L235A mutation and a P329G mutation.

7. The anti-IL1RAP antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, comprising a light chain constant region, wherein the light chain constant region is a Lambda or Kappa light chain constant region, such as a human Lambda or Kappa light chain constant region, preferably, the light chain constant region (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 53; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO:

53.

8. The anti-IL1RAP antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, comprising a heavy chain, wherein the heavy chain (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 32-36; or (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 32-36; and / or The antibody or antigen-binding fragment thereof comprises a light chain, wherein the light chain (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 42-44; or (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 42-44.

9. An anti-IL1RAP antibody or an antigen-binding fragment thereof, comprising a heavy chain and a light chain, wherein (i) the heavy chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 35 or 36, and The light chain comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 44; (ii) the heavy chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:32, and the light chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:42; or (iii) the heavy chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 33 or 34, and the light chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:

43.

10. The anti-IL1RAP antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, comprising a heavy chain and a light chain, wherein (i) the heavy chain comprises the amino acid sequence of SEQ ID NO: 35 or 36, and the light chain comprises the amino acid sequence of SEQ ID NO: 44; (ii) the heavy chain consists of the amino acid sequence of SEQ ID NO: 35 or 36, and the light chain consists of the amino acid sequence of SEQ ID NO: 44; (iii) the heavy chain comprises the amino acid sequence of SEQ ID NO: 32, and the light chain comprises the amino acid sequence of SEQ ID NO: 42; (iv) the heavy chain consists of the amino acid sequence of SEQ ID NO: 32, and the light chain consists of the amino acid sequence of SEQ ID NO: 42; (v) the heavy chain comprises the amino acid sequence of SEQ ID NO: 33 or 34, and the light chain comprises the amino acid sequence of SEQ ID NO: 43; or (vi) the heavy chain consists of the amino acid sequence of SEQ ID NO: 33 or 34, and the light chain consists of the amino acid sequence of SEQ ID NO:

43. 11 . The anti-IL1RAP antibody or antigen-binding fragment thereof according to claim 1 , wherein the antibody is a monoclonal antibody; and / or the antibody is a humanized antibody or a chimeric antibody.

12. The anti-IL1RAP antibody or antigen-binding fragment thereof of any one of claims 1 to 11, wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, dAb (domain antibody), diabody, or linear antibody.

13. The anti-IL1RAP antibody or antigen-binding fragment thereof of any one of claims 1-12, wherein the antibody or antigen-binding fragment thereof has one or more of the following properties: a) binds to IL1RAP (eg, human IL1RAP) with high affinity; b) Effectively blocks the binding of IL1α, IL1β, IL33, and IL36 to the IL1RAP receptor complex, thereby relieving the inhibitory effect on the downstream NF-kB signaling pathway; c) inhibiting IL-1 (e.g., IL1β), IL-36, and / or IL33-mediated signaling pathways, e.g., in epithelial cells or PBMCs; d) having good pharmacokinetic characteristics, such as good stability (e.g., colloidal stability and / or thermal stability), good solubility and / or long half-life; e) effectively preventing or treating inflammation, for example, peritoneal inflammation such as peritonitis (eg, acute peritonitis), gout (eg, acute gout) and / or skin inflammation (eg, dermatitis).

14. An isolated nucleic acid encoding the anti-IL1RAP antibody or antigen-binding fragment thereof of any one of claims 1-13.

15. A vector comprising the nucleic acid of claim 14, preferably said vector is an expression vector.

16. A host cell comprising the nucleic acid of claim 14 or the vector of claim 15, preferably, the host cell is prokaryotic or eukaryotic, more preferably selected from yeast cells, mammalian cells (e.g., 293 cells or CHO cells, e.g., CHO-K cells or HEK293 cells) or other cells suitable for preparing antibodies or antigen-binding fragments thereof.

17. A method for preparing an anti-IL1RAP antibody or an antigen-binding fragment thereof, the method comprising a) culturing the host cell of claim 16 under conditions suitable for expressing a nucleic acid encoding the anti-IL1RAP antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, b) optionally isolating said antibody or antigen-binding fragment thereof, c) Optionally, the method further comprises recovering the anti-IL1RAP antibody or antigen-binding fragment thereof from the host cell. Optionally, the antibody is purified, for example, by Protein A purification. An immunoconjugate comprising the anti-IL1RAP antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 and other substances, such as chemotherapeutic agents, toxins, small molecule drugs, cytotoxic agents, apoptotic agents, chelating agents, immunomodulators, such as immunosuppressants.

19. A pharmaceutical composition comprising the anti-IL1RAP antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 or the immunoconjugate according to claim 18, and optionally a pharmaceutically acceptable excipient.

20. A pharmaceutical combination comprising the anti-IL1RAP antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 or the immunoconjugate according to claim 18, and one or more other therapeutic agents, for example, the therapeutic agent is selected from cytokines, other antibodies, small molecule drugs or immunomodulators (e.g., immunosuppressants).

21. A method for preventing or treating a disease or condition associated with inappropriate activation of a pathway mediated by an IL1RAP receptor complex in an individual, the method comprising administering to the subject an effective amount of the anti-IL1RAP antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, or the immunoconjugate according to claim 18, or the pharmaceutical composition according to claim 19, or the pharmaceutical combination according to claim 20, for example, the disease or condition is selected from a tumor such as cancer, or inflammation, such as peritoneal inflammation such as peritonitis (e.g., acute peritonitis), gout (e.g., acute gout), and / or skin inflammation (e.g., dermatitis); Optionally, the subject has abnormal activation of a signaling pathway mediated by the IL1RAP receptor complex compared to healthy individuals, Optionally, the method further comprises administering one or more other therapies, such as treatment modalities and / or other therapeutic agents, for example, selected from cytokines, other antibodies, small molecule drugs, or immunomodulators (eg, immunosuppressants).

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