Glucocorticoid drug conjugates

Abstract

An antibody-drug conjugate represented by formula (I), wherein Ab is an antibody or an antigen-binding fragment thereof, L is a linker covalently linking Ab to D, and k is 1 to 20; and D is a glucocorticoid or a residue thereof, wherein the glucocorticoid is selected from budesonide or ciclesonide. TIFF2025508665000103.tif11150

Description

[Technical field]

[0001] The present disclosure is in the pharmaceutical field and specifically relates to glucocorticoid drug conjugates. [Background technology]

[0002] Interleukin-4 (IL-4) consists of 153 amino acids and has a molecular weight of about 17 kDa. IL-4 was first discovered because it can stimulate B cell proliferation, and was named B cell stimulatory factor-1 (BSF-1). IL-4, like IL-13, belongs to the type I cytokine family and has a quaternary structure composed of a hydrophobic bundle core of four α-helices. IL-4 is secreted by TH2 cells, participates in TH2-mediated immune responses, and has a wide range of biological activities, including stimulating the proliferation of T cells, mast cells, granulocytes, megakaryocytes, and erythrocytes. In addition, IL-4 can also stimulate B cells to express major histocompatibility complex class 2 molecules. IL-13 has about 30% amino acid sequence homology to IL-4 and multiple similar functions. Both IL-4 and IL-13 can promote B cell proliferation and induce IgM-type IgE conversion in combination with CD40 / CD40L costimulation. IL-4 promotes mast cell aggregation, upregulates the expression of high affinity IgE receptors on mast cells and low affinity IgE receptor CD23 (FcεRII) on B cells, upregulates the expression of vascular endothelial cell adhesion molecule (VCAM-1), and promotes the migration of eosinophils, T lymphocytes, monocytes, and basophils. Unlike IL-13, IL-4 can promote the differentiation of naive T cells into TH2.

[0003] IL-4 needs to bind to a membrane receptor to exert its biological function. Human interleukin receptor (IL-4R) is a heterodimer formed by two polypeptide chains, one of which, the α chain, has a very high affinity for IL-4, and the IL-4Rα chain plays a leading role in binding to IL-4 in the IL-4R complex, so IL-4Rα is often used instead of IL-4R in many scientific studies and reports. IL-4R is expressed in various cells, such as human B cells, mast cells, eosinophils, basophils, macrophages / monocytes, DC cells, fibrocytes, airway epithelium and smooth muscle. IL-4Rα can form two types of receptor complexes with other subunits, and in hematopoietic stem cells it mainly expresses type I receptors consisting of IL-4Rα and γc. In non-hematopoietic stem cells, IL-4 acts mainly through type II receptors consisting of IL-4Rα and IL-13Rα1. Type II receptor is a co-receptor for IL-4 and IL-13, and IL-13 functions by binding to IL-13Rα1. Both type I and type II receptors transmit signals through the Jak / STAT pathway, and IL-4Rα, γc, and IL-13Rα1 bind to Jak1, Jak3, and Tyk2, respectively, to activate downstream pathways, and IL-4 and IL-13 can also transmit signals through the insulin receptor substrate family (IRS) to finally activate PI3-K and NF-κB in the nucleus. Blocking IL-4R can inhibit the biological functions of not only IL-4 but also IL-13.

[0004] Several studies have shown that IL-4 and IL-13 are linked to diseases associated with TH2 immune responses. Atopic dermatitis (AD), also known as atopic eczema or hereditary allergic dermatitis, is a common dermatological disease that is prevalent in children and adolescents and often occurs together with certain hereditary allergic diseases such as allergic rhinitis and asthma. Studies have found that AD patients have elevated levels of TH2 factors IL-4, IL-5, IL-10, and IL-13, as well as elevated IgE levels, and further found that TH2 factors are associated with AD disease progression, and that mice overexpressing TH2 factors such as IL-4 and IL-13 exhibit defective skin protection and AD-like pathology.

[14]

[15] Increased levels of IL-4 and IL-13 in AD patients inhibit epithelial differentiation and the production of antimicrobial peptides. IL-4-deficient mice have reduced incidence of allergic inflammation in the skin. These studies indicate that blocking IL-4R may be effective in treating AD. Anti-IL-4R monoclonal antibodies are already on the market overseas and show good therapeutic effects against AD.

[0005] IL-13 and IL-4 also play important roles in asthma. Asthma is a common inflammatory lung disease characterized by airway hyperresponsiveness (AHR), mucus hypersecretion, fibrosis and elevated IgE levels. Non-specific stimuli such as cold air often lead to exacerbation of airway hyperresponsiveness, and AHR and mucus hypersecretion lead to airway obstruction, which is the main cause of asthma death. TH2 factors play an important role in the progression of asthma disease, and IL-4 and IL-13 are overexpressed in bronchial and alveolar lavage fluids of asthmatic patients. Although IL-13 and IL-4 have some functional similarities, several studies have shown that IL-13 plays a more important role in the progression of asthma disease than other Th2 cytokines. IL-13 can promote goblet cell differentiation and fibrosis. Injection of recombinant IL-13 into the airways of mice not stimulated with allergens results in airway inflammation, mucus hypersecretion and airway hyperresponsiveness, and injection of soluble IL13Rα2 can block the development of AHR, mucus hypersecretion and lung inflammation in mice. Injection of IL-4Rα antibodies in an asthma model can reduce AHR and eosinophils in alveolar lavage fluid. Studies indicate that blocking IL-4Rα can be effective in treating asthma.

[0006] Currently, many pharmaceutical companies around the world are working on developing monoclonal antibodies against IL-4R, and related patent applications include, for example, WO2010053751, WO2001092340, WO2008054606, WO2014031610, and WO2020038454.

[0007] Glucocorticoids are also relatively effective drugs for treating allergic diseases, inflammation, etc. Representative glucocorticoids include glucocorticoids produced in vivo, such as cortisol and corticosterone, and synthetic glucocorticoids, such as dexamethasone, prednisone, prednisolone, and budesonide. These glucocorticoids have a steroid structure, and are collectively called steroids, and are used in the treatment of various diseases. However, the use of these steroids may cause side effects, such as steroid peptic ulcer, steroid purpura, steroid pancreatitis, steroid diabetes, steroid cataract, and steroid glaucoma.

[0008] Antibody drug conjugates (ADCs) refer to monoclonal antibodies or antibody fragments linked to biologically active drugs via stable chemical linker compounds. Most ADCs in preclinical and clinical development are used for oncological indications, where cytotoxic payloads target antigen-expressing cancer cells. However, modulation of pathogenic cell activity by ADC-mediated delivery of biologically active small molecules is also attractive for non-oncological indications, leading to the widespread application of the technology.

[0009] Several glucocorticoid drug conjugates have been disclosed in the prior art, such as WO2017210471, WO2019106609, WO2019136487, etc. Summary of the Invention

[0010] One aspect of the present disclosure is a compound of formula (I): [ka] [In the formula, Ab is an anti-IL-4R antibody or an antigen-binding fragment thereof, L is a linker covalently linking Ab to D, and k is 1 to 20, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or any number between any two of these; D is a glucocorticoid or a residue thereof, the glucocorticoid being selected from dexamethasone, prednisone, prednisolone, budesonide, mometasone, beclomethasone dipropionate, fluticasone, triamcinolone acetonide and ciclesonide, and may be, for example, budesonide or ciclesonide. The present invention provides an antibody-drug conjugate (ADC) represented by the formula:

[0011] In some embodiments, D has the formula: [ka]

[0012] In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof may be a known antibody, for example, an anti-IL-4R antibody or antigen-binding fragment thereof described in WO2010053751, WO2001092340, WO2008054606, WO2014031610, or WO2020038454 (each of which is incorporated herein by reference).

[0013] In some embodiments, anti-IL-4R antibodies or antigen-binding fragments thereof include, but are not limited to, Dupixent, PRS-060, AK-120, 63 IgG1, CBP201, AMG-317, or antigen-binding fragments thereof.

[0014] In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof is an anti-human IL-4R antibody or antigen-binding fragment thereof.

[0015] In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: (I) HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively; or (II) HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, respectively; and / or the antibody light chain variable region (I) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively; or (II) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively; or (III) LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 38, SEQ ID NO: 7 and SEQ ID NO: 40, respectively; or (IV) comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 42, SEQ ID NO: 39 and SEQ ID NO: 8, respectively.

[0016] Table 1 shows the CDR sequences of anti-IL-4R antibodies or antigen-binding fragments thereof. [Table 1]

[0017] In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof is selected from the group consisting of the following (I) to (IV): (I) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, respectively; (II) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively; (III) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO:38, SEQ ID NO:7 and SEQ ID NO:40, respectively; (IV) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO:42, SEQ ID NO:39 and SEQ ID NO:8, respectively; Includes one selected from:

[0018] In some embodiments, the anti-IL-4R antibody or antigen-binding fragment is a murine antibody, a chimeric antibody, a fully human antibody, a humanized antibody or a fragment thereof. In some specific embodiments, the anti-IL-4R antibody or antigen-binding fragment is humanized.

[0019] In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof comprises a FR region sequence derived from human germline light chain IGKV3-11*01 (SEQ ID NO: 22, for antibody 25G7) or a sequence with back mutations having at least 95% identity thereto. In some specific embodiments, the back mutations are one or more selected from L46P, L47W, and F71Y. In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof comprises a FR region sequence derived from human germline heavy chain IGHV3-48*01 (SEQ ID NO: 21, for antibody 25G7) or a sequence with back mutations having at least 95% identity thereto. In some specific embodiments, the back mutations are one or more selected from S94A, F67S, and A93T. In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof comprises a FR region sequence derived from human germline light chain IGKV2D-29*01 (SEQ ID NO: 24, for antibody 7B10) or a backmutation sequence having at least 95% identity thereto. In some specific embodiments, the backmutation is selected from M4L and / or V58I. In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof comprises a FR region sequence derived from human germline heavy chain IGHV1-2*02 (SEQ ID NO: 23, for antibody 7B10) or a backmutation sequence having at least 95% identity thereto. In some specific embodiments, the backmutation is one or more selected from M69L, R71I, T73K, R94K.

[0020] Human germline heavy chain IGHV3-48*01: TIFF2025508665000004.tif27155

[0021] Human germline light chain IGKV3-11*01: TIFF2025508665000005.tif27155

[0022] Human germline heavy chain IGHV1-2*02: TIFF2025508665000006.tif27155

[0023] Human germline light chain IGKV2D-29*01: TIFF2025508665000007.tif27155

[0024] In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, The heavy chain variable region is (I) a sequence as set forth in SEQ ID NO:1 or having at least 70%, 80%, 90%, 95%, 98%, 99% identity to SEQ ID NO:1; or (II) a sequence set forth in SEQ ID NO:9 or having at least 70%, 80%, 90%, 95%, 98%, 99% identity to SEQ ID NO:9; or (III) a sequence set forth in SEQ ID NO: 43 or having at least 70%, 80%, 90%, 95%, 98%, 99% identity to SEQ ID NO: 43; or and / or the light chain variable region (I) a sequence as set forth in SEQ ID NO:2 or having at least 70%, 80%, 90%, 95%, 98%, 99% identity to SEQ ID NO:2; or (II) a sequence set forth in SEQ ID NO:10 or having at least 70%, 80%, 90%, 95%, 98%, 99% identity to SEQ ID NO:10; or (III) a sequence as set forth in SEQ ID NO: 37 or having at least 70%, 80%, 90%, 95%, 98%, 99% identity to SEQ ID NO: 37; or (IV) A sequence set forth in SEQ ID NO: 41 or having at least 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 41.

[0025] 25G7 HCVR (25G7 heavy chain variable region) TIFF2025508665000008.tif32155

[0026] 25G7 LCVR (25G7 light chain variable region) TIFF2025508665000009.tif27155

[0027] 7B10 HCVR (7B10 heavy chain variable region) TIFF2025508665000010.tif32155

[0028] 7B10 LCVR (7B10 light chain variable region) TIFF2025508665000011.tif32155

[0029] hu25G7-A LCVR (hu25G7-A light chain variable region) TIFF2025508665000012.tif27155

[0030] hu25G7-B LCVR (hu25G7-B light chain variable region) TIFF2025508665000013.tif27155

[0031] hu25G7-VH (hu25G7 heavy chain variable region) TIFF2025508665000014.tif32155

[0032] In at least one embodiment, the anti-IL-4R antibody or antigen-binding fragment has a heavy chain variable region represented by the sequence of SEQ ID NO:1 and a light chain variable region represented by the sequence of SEQ ID NO:2; or the heavy chain variable region is represented by the sequence SEQ ID NO: 9 and the light chain variable region is represented by the sequence SEQ ID NO: 10; or the heavy chain variable region is represented by the sequence SEQ ID NO: 43 and the light chain variable region is represented by the sequence SEQ ID NO: 37; or The heavy chain variable region is shown in the sequence SEQ ID NO:43, and the light chain variable region is shown in the sequence SEQ ID NO:41.

[0033] In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, The heavy chain variable region is (I) a sequence represented by one of SEQ ID NOs: 25 to 27, or a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to one of SEQ ID NOs: 25 to 27, or (II) a sequence represented by one of SEQ ID NOs: 31 to 33, or a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to one of SEQ ID NOs: 31 to 33; and / or the light chain variable region (I) a sequence represented by one of SEQ ID NOs: 28 to 30, or a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to one of SEQ ID NOs: 28 to 30, or (II) A sequence represented by one of SEQ ID NOs: 34 to 36, or a sequence having at least 70%, 80%, 90%, 95%, 98% or 99% identity to one of SEQ ID NOs: 34 to 36.

[0034] hu25G7-VH-a: TIFF2025508665000015.tif32155

[0035] hu25G7-VH-b: TIFF2025508665000016.tif32155

[0036] hu25G7-VH-c: TIFF2025508665000017.tif32155

[0037] hu25G7-VL-a: TIFF2025508665000018.tif27155

[0038] hu25G7-VL-b: TIFF2025508665000019.tif27155

[0039] hu25G7-VL-c: TIFF2025508665000020.tif27155

[0040] hu7B10-VH-a: TIFF2025508665000021.tif32155

[0041] hu7B10-VH-b: TIFF2025508665000022.tif32155

[0042] hu7B10-VH-c: TIFF2025508665000023.tif32155

[0043] hu7B10-VL-a: TIFF2025508665000024.tif32155

[0044] hu7B10-VL-b: TIFF2025508665000025.tif32155

[0045] hu7B10-VL-c: TIFF2025508665000026.tif32155

[0046] In some specific embodiments, the heavy chain variable region is represented by one of the sequences set forth in SEQ ID NOs: 25-27, and the light chain variable region is represented by one of the sequences set forth in SEQ ID NOs: 28-30; The heavy chain variable region is represented by one of the sequences set forth in SEQ ID NOs: 31-33, and the light chain variable region is represented by one of the sequences set forth in SEQ ID NOs: 34-36.

[0047] In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof comprises a heavy chain constant region selected from human IgG1, IgG2, IgG3, or IgG4, or a variant thereof. In some specific embodiments, the heavy chain constant region comprises a human IgG1 heavy chain constant region or a variant thereof. In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof comprises a constant region of a human κ or λ chain, or a variant thereof.

[0048] In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof is a humanized antibody, wherein the heavy chain sequence is set forth in SEQ ID NO: 17 or has at least 85% sequence identity thereto, and the light chain sequence is set forth in SEQ ID NO: 18 or has at least 85% sequence identity thereto.

[0049] In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof is a humanized antibody, wherein the heavy chain sequence is set forth in SEQ ID NO: 19 or has at least 85% sequence identity thereto, and the light chain sequence is set forth in SEQ ID NO: 20 or has at least 85% sequence identity thereto.

[0050] In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof is a humanized antibody, wherein the heavy chain sequence is set forth in SEQ ID NO:44 or has at least 85% sequence identity thereto, and the light chain sequence is set forth in SEQ ID NO:45 or has at least 85% sequence identity thereto.

[0051] In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof is a humanized antibody, wherein the heavy chain sequence is set forth in SEQ ID NO:44 or has at least 85% sequence identity thereto, and the light chain sequence is set forth in SEQ ID NO:46 or has at least 85% sequence identity thereto.

[0052] In some embodiments, the present invention provides an isolated anti-IL-4R antibody or antigen-binding fragment thereof, which binds to human IL-4R or an epitope thereof in competition with any one of the above-mentioned anti-IL-4R antibodies or antigen-binding fragments thereof.

[0053] In some embodiments, a bispecific or multispecific antibody is provided that comprises the light chain variable region and / or the heavy chain variable region of any one of the above-mentioned anti-IL-4R antibodies or antigen-binding fragments thereof. In other embodiments, a single-chain antibody is provided that comprises the light chain variable region and / or the heavy chain variable region of any one of the above-mentioned anti-IL-4R antibodies or antigen-binding fragments thereof.

[0054] In some embodiments, the humanized anti-IL-4R antibody or antigen-binding fragment thereof further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4, or a variant thereof. In at least one embodiment, a human IgG2 or IgG4 heavy chain constant region is included. This is because IgG2 and IgG4 do not have ADCC toxicity. In another embodiment, an IgG1 is used that does not have ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutation. In at least one embodiment, the variant comprises a heavy chain constant region mutation that reduces or eliminates ADCC effector function, such as, but not limited to, N297A, L234A, and L235A of IgG1. In some embodiments, the IgG1 comprises mutations E239D and M241L.

[0055] Unless otherwise specified, the anti-IL-4R antibodies or antigen-binding proteins thereof of the present disclosure are encoded by Kabat.

[0056] In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises: (I) a sequence as set forth in SEQ ID NO: 17 or having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 17; or (II) a sequence set forth in SEQ ID NO: 19 or having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 19; or (III) a sequence as set forth in SEQ ID NO: 44 or having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 44; (IV) a sequence set forth in SEQ ID NO: 47 or having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 47; and / or the light chain (I) a sequence as set forth in SEQ ID NO: 18 or having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 18; or (II) a sequence set forth in SEQ ID NO:20 or having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO:20; or (III) a sequence as set forth in SEQ ID NO: 45 or having at least 90%, 95%, 98% or 99% identity to SEQ ID NO: 45; or (IV) A sequence set forth in SEQ ID NO: 46 or having at least 90%, 95%, 98% or 99% identity to SEQ ID NO: 46.

[0057] hu25G7 HC TIFF2025508665000027.tif80155

[0058] hu25G7 LC TIFF2025508665000028.tif43155

[0059] hu7B10HC TIFF2025508665000029.tif80155

[0060] hu7B10 LC TIFF2025508665000030.tif48155

[0061] hu25G7-IgG4 HC TIFF2025508665000031.tif80155

[0062] hu25G7-A LC TIFF2025508665000032.tif43155

[0063] hu25G7-B LC TIFF2025508665000033.tif43155

[0064] hu25G7-24-IgG1(E239D,M241L)HC TIFF2025508665000034.tif80155

[0065] In some embodiments, the heavy chain sequence is set forth in SEQ ID NO: 17 and the light chain sequence is set forth in SEQ ID NO: 18; or the heavy chain sequence is shown in SEQ ID NO: 19 and the light chain sequence is shown in SEQ ID NO: 20; or the heavy chain sequence is shown in SEQ ID NO: 44 and the light chain sequence is shown in SEQ ID NO: 45; or the heavy chain sequence is shown in SEQ ID NO: 44 and the light chain sequence is shown in SEQ ID NO: 46; or The heavy chain sequence is shown in SEQ ID NO:47 and the light chain sequence is shown in SEQ ID NO:45.

[0066] In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises: (I) a sequence as set forth in SEQ ID NO: 48 or having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 48; or (II) a sequence set forth in SEQ ID NO: 49 or having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 49; or (III) a sequence as set forth in SEQ ID NO: 50 or having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 50; (IV) a sequence set forth in SEQ ID NO:51 or having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO:51; and / or the light chain (I) a sequence as set forth in SEQ ID NO: 18 or having at least 70%, 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 18; or (II) a sequence as set forth in SEQ ID NO: 45 or having at least 90%, 95%, 98% or 99% identity to SEQ ID NO: 45; or (III) A sequence set forth in SEQ ID NO: 46 or having at least 90%, 95%, 98% or 99% identity to SEQ ID NO: 46.

[0067] hu25G7-IgG4 VH-CH1 TIFF2025508665000035.tif43155

[0068] hu25G7-IgG1 VH-CH1 TIFF2025508665000036.tif43155

[0069] hu25G7-24-IgG4 VH-CH1 TIFF2025508665000037.tif43155

[0070] hu25G7-24-IgG1 VH-CH1 TIFF2025508665000038.tif43155

[0071] In some embodiments, the heavy chain sequence is set forth in SEQ ID NO:48 and the light chain sequence is set forth in SEQ ID NO:18; or the heavy chain sequence is shown in SEQ ID NO:49 and the light chain sequence is shown in SEQ ID NO:18; or the heavy chain sequence is shown in SEQ ID NO:50 and the light chain sequence is shown in SEQ ID NO:45; or the heavy chain sequence is shown in SEQ ID NO:50 and the light chain sequence is shown in SEQ ID NO:46; or the heavy chain sequence is shown in SEQ ID NO:51 and the light chain sequence is shown in SEQ ID NO:45; or The heavy chain sequence is shown in SEQ ID NO:51 and the light chain sequence is shown in SEQ ID NO:46.

[0072] In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof may be an antibody variant, the variant having 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid changes in the light chain and / or 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid changes in the heavy chain.

[0073] In some embodiments, the variant has the same or similar biological function or effect as the parent anti-IL-4R antibody or fragment thereof.

[0074] In some embodiments, the antigen-binding fragment includes, but is not limited to, a Fab, a Fab', an Fv, a F(ab')2, a linear antibody, a scFv (single chain Fv antibody), a tandem di-scFv, a tandem tri-scFv, a diabody, a triabody, a tetrabody, a sdAb (single domain antibody or nanobody), a sdFv, a peptibody, a domain antibody, a multispecific antibody (e.g., a bispecific antibody, a triabody, or a tetraspecific antibody), a dsFv (a disulfide bond stabilized Fv), a ScdsFv (a disulfide bond stabilized single chain Fv antibody).

[0075] In some embodiments, the antigen-binding fragment of an anti-IL-4R antibody in the antibody-drug conjugate or a pharma- ceutically acceptable salt or solvate thereof of the present disclosure binds to the same IL-4R or epitope thereof as the antigen-binding fragment of the anti-IL-4R antibody described above.

[0076] In some embodiments, a polynucleotide, such as DNA or RNA, encoding the above-mentioned anti-IL-4R antibody or antigen-binding fragment thereof is provided.

[0077] In some embodiments, an expression vector, such as a eukaryotic expression vector, a prokaryotic expression vector, or a viral vector, is provided that comprises the polynucleotide.

[0078] In some embodiments, the present invention provides a host cell, e.g., a prokaryotic cell, or a eukaryotic cell, transformed with the expression vector. In some specific embodiments, the host cell is a bacterium (e.g., Escherichia coli), a yeast cell (e.g., Pichia spp.), or a mammalian cell (e.g., a Chinese hamster ovary (CHO) cell or a human embryonic kidney (HEK) 293 cell).

[0079] In some embodiments, a method of preparing the anti-IL-4R antibody or antigen-binding fragment thereof is provided, comprising expressing the antibody or antigen-binding fragment thereof in a host cell as described above, and isolating the antibody or antigen-binding fragment thereof from the host cell.

[0080] The three-letter and one-letter amino acid codes for the anti-IL-4R antibodies or antigen-binding fragments thereof disclosed herein are as described in J. Biol. Chem, 243, p. 3558 (1968).

[0081] In one embodiment, k is an arbitrary number between 1 and 10, and preferably an arbitrary number between 2 and 5. k may be an integer or a decimal number.

[0082] In certain embodiments, the linker is stable extracellularly, allowing it to remain intact when the ADC is in the extracellular environment, but is capable of being cleaved upon internalization into a cell. In some embodiments, when the ADC enters a cell that expresses an antigen specific for the antibody portion of the ADC, the glucocorticoid drug moiety is cleaved from the antibody portion, and cleavage releases the unmodified form of the glucocorticoid.

[0083] In some embodiments, the cleavable moiety in the linker is a cleavable peptide moiety. In some embodiments, ADCs comprising a cleavable peptide moiety exhibit lower aggregation levels, improved drug-antibody ratios, relative to ADCs comprising other cleavable moieties. In some embodiments, the addition of a cleavable moiety increases cytotoxicity and / or efficacy relative to non-cleavable linkers. In some embodiments, the cleavable peptide moiety can be cleaved by an enzyme, and the linker is an enzyme-cleavable linker. In some embodiments, the enzyme is a cathepsin, and the linker is a cathepsin-cleavable linker. In some embodiments, compared to other cleavage mechanisms, an enzyme-cleavable linker (e.g., a cathepsin-cleavable linker) exhibits one or more of the improved properties described above.

[0084] In some embodiments, the linker comprises a stretch unit, which is a chemical structural fragment that is covalently attached to the antibody via a carbon atom at one end and linked to an amino acid unit, a disulfide moiety, a sulfonamide moiety, or a non-peptide chemical moiety at the other end. Exemplary stretch units are: [ka] Including, but not limited to:

[0085] In some embodiments, the linker comprises an amino acid unit, the amino acid unit being phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, aspartic acid, homolysine, n-methyl-valine, [ka] (q is an integer of 1 to 6), and exemplary amino acid units include valine-citrulline (Val-Cit), alanine-phenylalanine (Ala-Phe), phenylalanine-lysine (Phe-Lys), phenylalanine-homolysine (Phe-Homolys), n-methyl-valine-citrulline (Me-Val-Cit), alanine-alanine (Ala-Ala), glycine-glutamic acid (Gly-Glu), glutamic acid-alanine-alanine (Glu-Ala-Ala), and glycine-lysine (Gly-Lys), glycine-valine-citrulline (Glv-Val-Cit), and glycine-glycine-glycine (Gly-Gly-Gly), [ka] Including, but not limited to:

[0086] In some embodiments, the linker may include at least one polyethylene glycol (PEG) moiety. The PEG moiety may be, for example, -(PEG)p1-, [ka] In which p1 is an integer from 1 to 20, for example, [ka] , (PEG)2, [ka] , (PEG)4, [ka] , (PEG)5 It is.

[0087] In some embodiments, the linker comprises a spacer unit linking D. In some embodiments, the spacer unit is p-aminobenzyloxycarbonyl (PAB), [ka] Includes.

[0088] In some embodiments, the Spacer unit is p-aminobenzoyl, [ka] Includes.

[0089] In some embodiments, the spacer unit is -NH(CH2) n1 -L a -L b -L c -, wherein n1 is an integer selected from 0 to 6, and L a -C(=O)-NH-, -NR 7 -(CH2) n2 -, -O- or a single bond; R 7 is hydrogen, C 1-6 Alkyl group, -(CH2) n3 -COOH, -(CH2) n4 n3 is an integer selected from 1 to 4; n4 is an integer selected from 1 to 6; L b HA-CR 8 (R 9 )-, -O-, -NR 10 - or a single bond, R 8 and R 9 are independently hydrogen, C 1-6 Alkyl group, C 3-6 Cycloalkyl groups, -(CH2) n5 -NH2, -(CH2) n6 -COOH, -(CH2) n7 -OH, R 10 is hydrogen or C 1-6 alkyl group, n5 is an integer selected from 0 to 6, n6 is an integer selected from 1 to 4, and n7 is an integer selected from 1 to 4, and when n5 is 0, R 8 and R 9 is different, or R 8 and R9 C together with the carbon atom connected to it 3-6 Forming a cycloalkyl group, L c represents -CH2- or -C(=O)-.

[0090] In some embodiments, -NH(CH) n1 -L a -L b -L c -L in c indicates -NHCH2-.

[0091] In some embodiments, -NH(CH) n1 -L a -L b -L c -R in 8 and R 9 are independently hydrogen, C 1-6 Alkyl group, C 3-6 It is selected from cycloalkyl groups, for example hydrogen, methyl, ethyl or cyclopropyl.

[0092] In some embodiments, -NH(CH) n1 -L a -L b -L c -L in a represents -O- or a single bond, L b HA-CR 8 (R 9 )- or a single bond; L c indicates -C(=O)-, R 8 and R 9 C together with the carbon atom connected to it 3-6 It forms a cycloalkyl group.

[0093] In some embodiments, -NH(CH) n1 -L a -L b -L c -L in a represents -O- or a single bond, L b HA-CR 8 (R 9 )- or a single bond; L cindicates -C(=O)-, R 8 and R 9 are independently hydrogen, C 1-6 Alkyl group, C 3-6 Cycloalkyl groups, -(CH2) n5 -NH2, -(CH2) n6 -COOH, -(CH2) n7 -OH, R 10 is hydrogen or C 1-6 alkyl group, n5 is an integer selected from 0 to 6, n6 is an integer selected from 1 to 4, and n7 is an integer selected from 1 to 4, and when n5 is 0, R 8 and R 9 is different.

[0094] In some embodiments, -NH(CH) n1 -L a -L b -L c -L in a represents -O- or a single bond, L b HA-CR 8 (R 9 )- or a single bond; L c indicates -CH2-.

[0095] In some embodiments, -NH(CH) n1 -L a -L b -L c -L in a represents -O- or a single bond, L b HA-CR 8 (R 9 )- or a single bond; L c represents -CH2-, and R 8 and R 9 are independently hydrogen, C 1-6 Alkyl group, C 3-6 It is selected from cycloalkyl groups, preferably hydrogen, methyl, ethyl or cyclopropyl.

[0096] In some embodiments, the -NH(CH) in the antibody-drug conjugate n1 -L a -Lb -L c - is -NHCH2-, -NHCH2CH2-, -NHCH2CH2CH2-, -NHCH2-O-CH2-, -NHCH2CH2-O-CH2-, -NH(CH2)3-C(O)-, -NHCH2-O-CH2-C(O)-, -NH(CH2)2-O-CH2-C(O)-, [ka] It is.

[0097] In some embodiments, -NH-(CH) n1 -L a -L b -L c - is -NHCH2- or -NHCH2CH2CH2-.

[0098] In some embodiments, the spacer unit in the linker comprises (PEG)2. In some embodiments, although the linker is relatively short in length, ADCs comprising a relatively short spacer unit (e.g., (PEG)2) exhibit lower aggregation levels and / or higher drug loading relative to ADCs comprising a relatively long spacer unit (e.g., (PEG)8).

[0099] In another embodiment, the LD in an antibody conjugate (ADC) of the present disclosure has the formula: [ka] [wherein Str is a stretch unit covalently attached to Ab, Sp is a spacer unit, Pep may be an amino acid unit, a disulfide moiety, a sulfonamide moiety or the following non-peptide chemical moieties: [ka] wherein W is -NH-heterocycloalkylene- or heterocycloalkyl group, and Y is a heteroarylene group, an arylene group, -C(O)C 1-6Alkylene group, C 2-6 Alkenylene group, C 1-6 Alkylene group or -C 1-6 alkylene-NH-, and each R 16 is independently C 1-6 Alkyl group, C 2-6 Alkenyl group, -(C 1-6 alkylene)NHC(NH)NH2 or -(C 1-6 alkylene)NHC(O)NH2; R 17 and R 18 are each independently hydrogen, C 1-6 Alkyl group, C 2-6 an alkenyl group, an aryl group, a heteroaryl group, or R 17 and R 18 Let's go together 3-6 A cycloalkyl group can be formed, R 19 and R 20 are each independently C 1-6 Alkyl group, C 2-6 Alkenyl groups, aryl groups, heteroaryl groups, (C 1-6 alkyl)OCH2-, or R 19 and R 20 Let's go together 3-6 can form a cycloalkyl ring. It is a chemical moiety represented by the formula:

[0100] In some embodiments, Str in the antibody-drug conjugate (ADC) is [ka] Among these, R 21 is -W1-C(O)-, -C(O)-W1-C(O)-, -(CH2CH2O) p1 C(O)-, -(CH2CH2O) p1 CH2C(O)-, -(CH2CH2O) p1 CH2CH2C(O)-, in which W1 is C 1-6 Alkylene group, C 1-6alkylene-cycloalkyl groups or linear heteroalkyl groups of 1 to 8 atoms, the heteroalkyl groups containing 1 to 3 heteroatoms selected from N, O or S, wherein the alkyl groups, cycloalkyl groups and linear heteroalkyl groups each independently optionally contain halogen, deuterium, hydroxyl group, cyano group, amino group, C 1-6 Alkyl group, haloC 1-6 Alkyl groups, deuterated C 1-6 Alkyl group, C 1-6 Alkoxy groups and C 3-6 cycloalkyl groups, each p1 independently represents an integer of 1 to 20; L 1 -NR 22 -(CH2CH2O) p2 CH2CH2C(O)-, -NR 22 -(CH2CH2O) p2 CH2C(O)-, -S(CH2) p2 C(O)-, -(CH2) p2 C(O)- or a single bond, preferably a single bond; p2 is an integer of 1 to 20; R 22 is a hydrogen atom, C 1-6 Alkyl group, haloC 1-6 Alkyl group or deuterated C 1-6 It is selected from alkyl groups.

[0101] In some embodiments, R in Str of the antibody-drug conjugate 21 is C 1-6 It is selected from alkyleneC(O)-, -(CH2-CH2O)2C(O)-, -(CH2-CH2O)2CH2C(O)-, -(CH2-CH2O)2CH2CH2C(O)-, -(CH2-CH2O)2CH2CH2C(O)-, -(CH2-CH2O)3C(O)- and -(CH2-CH2O)4C(O)-.

[0102] In some embodiments, the Pep is selected from valine-citrulline (Val-Cit), alanine-alanine-asparagine (Ala-Ala-Asn), glycine-glycine-lysine (Gly-Gly-lys), valine-lysine (Val-lys), valine-alanine (Val-Ala), valine-phenylalanine (Val-Phe), or glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly).

[0103] In some embodiments, Sp is -NHCH-, -NHCHCH-, -NHCHCHCH-, -NHCH-O-CH-, -NHCHCH-O-CH-, -NH(CH)-C(O)-, -NHCH-O-CH-C(O)-, -NH(CH)-O-CH-C(O)-, [ka] Selected from.

[0104] In some embodiments, the linker L in the antibody-drug conjugate is maleimide-(PEG)-CH2CH2C(O)-Gly-Gly-Phe-Gly, maleimide-(PEG)-Val-Cit, maleimide-(PEG)-6-Val-Cit, maleimide-(PEG)-Val-Cit, maleimide-(PEG)-CH2CH2C(O)-Val-lys, maleimide-(CH2)-Val-Cit, maleimide-(CH2)-Val-lys, maleimide-(CH2)-Gly-Gly-Phe-Gly, maleimide- Maleimide-(PEG)-Gly-Gly-Phe-Gly, maleimide-(PEG)-Ala-Ala-Asn, maleimide-(PEG)-Ala-Ala-Asn, maleimide-(PEG)-triazole-(PEG)-sulfonamide, maleimide-(PEG)-CH2CH2C(O)-Val-lys, maleimide-(PEG)-triazole-(PEG)-sulfonamide or Mal-(PEG)-triazole-(PEG)-disulfide.

[0105] In some embodiments, the antibody-drug conjugate (ADC) of the present disclosure has the formula: [ka] [In the formula, k is selected from 1 to 10 and may be an integer or a decimal point, p1 is selected from 2, 4, 6, or 8, and p3 and p4 are each independently selected from 0, 1, or 2] [ka] In the formula, k is selected from 1 to 10 and may be an integer or a decimal point; p1 is selected from 2, 4, 6 or 8; p3 and p4 are each independently selected from 0, 1 or 2.

[0106] In some embodiments, an antibody-drug conjugate (ADC) according to the present disclosure comprises: [ka] In this case, k is selected from 1 to 10 and may be an integer or a decimal number.

[0107] The present disclosure provides a compound of formula II-A: [ka] [In the formula, p1 is selected from 2, 4, 6, or 8, and p3 and p4 are each independently selected from 0, 1, or 2] or a medicamentable salt thereof.

[0108] The present disclosure provides compounds of formula II-B: [ka] [In the formula, X is a halogen; p1 is selected from 2, 4, 6, or 8; and p3 and p4 are each independently selected from 0, 1, or 2.] or a medicamentable salt thereof.

[0109] In the structures described in this disclosure, [ka] represents a single bond or a double bond.

[0110] The present disclosure further provides a pharmaceutical composition comprising at least one of the above-described antibody-drug conjugates and a pharma- ceutically acceptable vector, diluent or excipient.

[0111] In some embodiments, the unit dose of the pharmaceutical composition is 0.001 to 1000 mg.

[0112] In some embodiments, the pharmaceutical composition comprises, based on the total weight of the composition, 0.01% to 99.99% of the compound. In some embodiments, the pharmaceutical composition comprises 0.1% to 99.9% of the compound. In some embodiments, the pharmaceutical composition comprises 0.5% to 99.5% of the compound. In some embodiments, the pharmaceutical composition comprises 1% to 99% of the compound. In some embodiments, the pharmaceutical composition comprises 2% to 98% of the compound.

[0113] In some embodiments, based on the total weight of the composition, the pharmaceutical composition comprises 0.01%-99.99% of a pharma- ceutically acceptable vector, diluent, or excipient. In some embodiments, the pharmaceutical composition comprises 0.1%-99.9% of a pharma- ceutically acceptable vector, diluent, or excipient. In some embodiments, the pharmaceutical composition comprises 0.5%-99.5% of a pharma- ceutically acceptable vector, diluent, or excipient. In some embodiments, the pharmaceutical composition comprises 1%-99% of a pharma- ceutically acceptable vector, diluent, or excipient. In some embodiments, the pharmaceutical composition comprises 2%-98% of a pharma- ceutically acceptable vector, diluent, or excipient.

[0114] The antibody-drug conjugates of the present disclosure can be administered to a patient in a manner appropriate for the indication, for example, parenterally, topically, or by inhalation. For injection, the antagonist can be administered, for example, via intra-articular, intravenous, intramuscular, intralesional, intraperitoneal, or subcutaneous routes, by bolus injection or continuous infusion. Local administration at the disease or injury is contemplated as transdermal delivery and sustained release from implants. Delivery by inhalation includes, for example, nasal or oral inhalation, use of a nebulizer, inhalation of the antagonist in aerosol form, and the like. Other options include eye drops, oral formulations including pills, syrups, tablets, or chewing gum, and topical formulations such as lotions, gels, sprays, and ointments.

[0115] The present disclosure provides a compound of formula (I): [ka] [In the formula, Ab is an anti-IL-4R antibody or an antigen-binding fragment thereof, L is a linker covalently linking Ab to D, and k is 1 to 20, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or any number between any two of these; D is a glucocorticoid or a residue thereof. The present invention further provides an inhalable pharmaceutical composition comprising an antibody-drug conjugate of the formula:

[0116] Selectable Ab, L, and D are as described above.

[0117] In some embodiments, the Ab is an anti-IL-4R Fab fragment.

[0118] The present disclosure further provides the use of the above-described antibody-drug conjugate and / or pharmaceutical composition comprising the antibody-drug conjugate in the preparation of a medicament for treating or preventing an IL-4R-mediated disease or condition.

[0119] The present disclosure further provides the use of the antibody-drug conjugate and / or pharmaceutical composition comprising the antibody-drug conjugate in the preparation of a medicament for treating or preventing an immune disease or condition selected from asthma, nasal polyps, chronic sinusitis, allergic skin disease, eosinophilic esophagitis, chronic obstructive pulmonary disease, allergic rhinitis, arthritis, inflammatory disease, allergic reaction, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis disease and kidney disease, preferably asthma or allergic skin disease.

[0120] The present disclosure further provides a method of delivering a glucocorticoid to a cell expressing IL-4R, the method comprising contacting a cell expressing IL-4R with an antibody-drug conjugate described in this disclosure.

[0121] The present disclosure further provides a reagent kit comprising the antibody-drug conjugate or pharmaceutical composition described in this disclosure.

[0122] Detailed Description of the Invention Unless otherwise limited, all technical and scientific terms used in this disclosure are consistent with those commonly understood by those skilled in the art. Although the present disclosure can be carried out or tested by any method and material similar or equivalent to those described in this disclosure, the present disclosure describes the preferred methods and materials. In describing and claiming the present disclosure, the following terms are used in accordance with the following definitions.

[0123] When trade names are used in this disclosure, applicants intend to include formulations of products bearing that trade name, non-patented drugs and active drug portions of products bearing that trade name.

[0124] Unless specifically stated to the contrary, terms used in the specification and claims have the following meanings.

[0125] The terms "linker", "linking unit", "linker unit", "linker" or "linking fragment" refer to a chemical fragment or bond that is linked at one end to a ligand and at the other end to an agent, and may be linked to another linker and then to an agent.

[0126] The linker may include one or more linker elements. Exemplary linker elements include 6-maleimidocaproyl (MC), maleimidopropionyl (MP), valine-citrulline (Val-Cit or vc), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), and N-succinimidyl 4-(2-pyridylthio)pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate (SMCC, also referred to herein as MCC), and N-succinimidyl (4-iodo-acetyl)aminobenzoate (SIAB), which are derived from coupling with linker reagents. The linker may include a stretch unit, a spacer unit, an amino acid unit, and an elongation unit. They can be synthesized by methods known in the art, for example, the methods described in US2005-0238649A1. The linker may be a "cleavable linker" that facilitates release of the drug in the cell. For example, an acid-labile linker (e.g., hydrazone), a protease-sensitive (e.g., peptidase-sensitive) linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Research 52:127-131 (1992); U.S. Patent No. 5,208,020) may be used.

[0127] The term "stretch unit" refers to a chemical structural fragment that is covalently attached at one end to an antibody via a carbon atom and at the other end is linked to an amino acid unit, a disulfide moiety, a sulfonamide moiety or a non-peptide chemical moiety.

[0128] The term "spacer unit" refers to a fragment of a bifunctional compound structure that can be used to couple an amino acid unit with a glucocorticoid to ultimately form an antibody-drug conjugate, and such a coupling method allows selective attachment of the glucocorticoid to the amino acid unit.

[0129] The term "amino acid" refers to an organic compound whose molecular structure contains an amino group and a carboxy group, and both the amino group and the carboxy group are directly linked to a -CH- structure. The general formula is H2NCHRCOOH, where R is H, a substituted or unsubstituted alkyl group, etc. Depending on the attachment position of the amino group to the carbon atom in the carboxylic acid, they can be divided into α, β, γ, δ, ε... amino acids. In the living world, amino acids that constitute natural proteins have a specific structural feature, namely, their amino groups are directly linked to the α-carbon atom, i.e., α-amino acids include glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, histidine, asparagine, glutamic acid, lysine, glutamine, methionine, arginine, serine, threonine, cysteine, proline, etc. An example of an unnatural amino acid is citrulline. As is well known to those skilled in the art, unnatural amino acids do not constitute natural proteins and therefore are not involved in the synthesis of antibodies in the present disclosure. The three-letter and one-letter codes for amino acids used in the present disclosure are as described in J. Biol. Chem, 243, p3558 (1968). [Table 2-1] [Table 2-2]

[0130] The term "antibody-drug conjugate" refers to a ligand linked to a biologically active drug by a stable linking unit. In the present disclosure, "antibody-drug conjugate" (ADC) refers to a monoclonal antibody or antibody fragment linked to a biologically active glucocorticoid by a stable linking unit, where the antibody or antibody fragment can be bound to a glucocorticoid molecule containing a linker through certain groups therein (e.g., interchain disulfide bonds).

[0131] The term "drug loading amount" refers to the average amount of drugs loaded on each antibody-drug conjugate molecule in an antibody-drug conjugate group, and may be expressed as a ratio of the amount of drug to the amount of antibody. The range of the drug loading amount may be 1 to 20, preferably 1 to 10, glucocorticoids (D) linked to each antibody (Ab). In an embodiment of the present disclosure, the drug loading amount is represented by k, which may be illustratively 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or an average value between any two numbers. It is preferably 1 to 10, and more preferably an average value of 1 to 8, or 2 to 8, or 2 to 7, or 3 to 8, or 3 to 7, or 3 to 6, or 4 to 7, or 4 to 6, or 4 to 5. Conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA testing, monoclonal antibody molecular size variant assay (CE-SDS) and HPLC can be used to specifically identify the average amount of drug in each ADC molecule after the coupling reaction.

[0132] The monoclonal antibody molecular size variant assay (CE-SDS) disclosed herein can quantitatively determine the purity of recombinant monoclonal antibody products based on molecular weight size under reducing and non-reducing conditions according to the capillary electrophoresis method (2015 edition of Chinese Pharmacopoeia 0542) by sodium dodecyl sulfate capillary electrophoresis (CE-SDS) ultraviolet detection method.

[0133] In one embodiment of the disclosure, the glucocorticoid is coupled to the N-terminal amino group and / or the ε-amino group of a lysine residue of the ligand via a linking unit, and typically the number of drug molecules that can be coupled to the antibody in a coupling reaction is less than the theoretical maximum.

[0134] The amount of antibody-drug conjugate loaded is (1) controlling the molar ratio of the linking reagent to the monoclonal antibody; (2) controlling reaction time and temperature; (3) selecting different reaction reagents; The amount of oxygen can be controlled by a number of methods, including but not limited to:

[0135] The term "antibody" refers to an immunoglobulin, which is a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. Immunoglobulins differ in amino acid composition and sequence order of the heavy chain constant region, and therefore their antigenicity also differs. Thus, immunoglobulins can be divided into five types, or immunoglobulin isotypes, IgM, IgD, IgG, IgA, and IgE, whose corresponding heavy chains are μ, δ, γ, α, and ε chains, respectively. Ig of the same type can be further divided into different subclasses depending on the amino acid composition of the hinge region and the number and position of the heavy chain disulfide bonds, for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are divided into κ or λ chains depending on the difference in the constant region. Each of the five types of Ig may have a κ or λ chain.

[0136] In the heavy and light chains of antibodies, the sequence of about 110 amino acids near the N-terminus is largely variable and forms the variable region (Fv region), while the remaining amino acid sequence near the C-terminus is relatively stable and forms the constant region. The variable region includes three hypervariable regions (HVR) and four framework regions (FR) whose sequences are relatively conservative. The three hypervariable regions determine the specificity of the antibody and are also called complementarity determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, which are arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3, and the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.

[0137] Antibodies of the present disclosure include murine antibodies, chimeric antibodies, humanized antibodies and fully human antibodies, with humanized antibodies and fully human antibodies being preferred.

[0138] The term "mouse antibody" in this disclosure refers to an antibody prepared in mice according to the knowledge and skill in the art, in which a particular antigen is injected into a test subject, and hybridomas expressing antibodies with the desired sequence or functional characteristics are isolated.

[0139] The term "chimeric antibody" refers to an antibody in which the variable region of a mouse antibody is fused with the constant region of a human antibody, and can reduce the immune response reaction induced by mouse antibodies. To prepare a chimeric antibody, first, a hybridoma secreting a mouse-specific monoclonal antibody is prepared, and then the variable region gene is cloned from the mouse hybridoma cell, and if necessary, the constant region gene of a human antibody is cloned, and the mouse variable region gene and the human constant region gene are linked to form a chimeric gene, which is then inserted into an expression vector, and finally the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic cell system.

[0140] The term "humanized antibody", also called CDR-grafted antibody, refers to an antibody produced by grafting mouse CDR sequences onto a human antibody variable region framework, i.e., the framework sequences of various human germline antibodies. This overcomes the heterologous reaction induced by chimeric antibodies having a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases containing germline antibody gene sequences or from disclosed references. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk / vbase) and Kabat, EA et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. To avoid a decrease in activity associated with a decrease in immunogenicity, the activity can be maintained by performing the least possible back mutations or reverse mutations on the human antibody variable region framework sequences. The humanized antibodies of the present disclosure further include phage-displayed humanized antibodies that have been affinity-matured against the CDRs. References further describing methods by which murine antibodies can be used for humanization include, for example, Queen et al., Proc., Natl. Acad. Sci. USA, 88, 2869, 1991, and the methods of Winter and coworkers [Jones et al., Nature, 321, 522 (1986); Riechmann et al., Nature, 332, 323-327 (1988); Verhoeyen et al., Science, 239, 1534 (1988)].

[0141] The term "fully human antibody", "fully human antibody" or "fully humanized antibody" is also called "fully human monoclonal antibody", in which both the variable and constant regions of the antibody are of human origin, and immunogenicity, toxicity and side effects are eliminated. The development of monoclonal antibodies has undergone four stages, namely mouse monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully human monoclonal antibodies, respectively. The present disclosure is a fully human monoclonal antibody. The technologies related to the preparation of fully human antibodies mainly include human hybridoma technology, EBV transformed B lymphocyte technology, phage display technology, transgenic mouse antibody preparation technology and single B cell antibody preparation technology.

[0142] The term "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that fragments of a full-length antibody can perform the antigen-binding function of the antibody. Examples of binding fragments included in "antigen-binding fragment" include (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VH and VL domains of a single arm of an antibody; (v) a single domain or dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341:544-546); and (vi) an isolated complementarity determining region (CDR), or (vii) a combination of two or more isolated CDRs, which can be optionally linked by a synthetic linker. In addition, Fv fragments are encoded by genes in which the two domains VL and VH are separated, but can be produced by recombinant techniques as a single protein chain in which the VL and VH regions pair to form a monovalent molecule by linking them with a synthetic linker (called single-chain Fv (scFv), see, for example, Bird et al. (1988) Science 242:423-426, and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:5879-5883). Such single-chain antibodies are also intended to be included in the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained by conventional techniques known to those skilled in the art, and the fragments are screened for functionality in the same way as complete antibodies. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of complete immunoglobulins. The antibodies may be of different isotypes, such as, for example, IgG (eg, IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0143] Fab is an antibody fragment with a molecular weight of approximately 50,000 that has antigen-binding activity and is obtained by treating an IgG antibody molecule with the protease papain (amino acid residue at position 224, which cleaves the H chain). Of these, approximately the N-terminal half of the H chain and the entire L chain are bound together by disulfide bonds.

[0144] F(ab')2 is an antibody fragment containing two Fab regions linked at the hinge, and has a molecular weight of approximately 100,000 and has antigen-binding activity, and is obtained by digesting the lower portion of the two disulfide bonds in the hinge region of IgG with the enzyme pepsin.

[0145] Fab' is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity, which can be obtained by cleaving the disulfide bond in the hinge region of the above-mentioned F(ab')2.

[0146] Furthermore, by inserting DNA encoding the Fab' fragment of the antibody into a prokaryotic or eukaryotic expression vector and introducing the vector into a prokaryote or eukaryote, the Fab' can be expressed to produce the above-mentioned Fab'.

[0147] The term "single chain antibody", "single chain Fv" or "scFv" is meant to include molecules of an antibody heavy chain variable domain (or region, VH) and an antibody light chain variable domain (or region, VL) linked by a linker. Such scFv molecules may have the general structure NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. A suitable prior art linker consists of a repeating GGGGS amino acid sequence or a variant thereof, for example a 1-4 repeat variant (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in the present disclosure are described in Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol.

[0148] The term "CDR" refers to one of the six main hypervariable regions in the variable domain of an antibody that mediate antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al., (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242). As used herein, the Kabat definition of CDR applies only to CDR1, CDR2 and CDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) of the light chain variable domain and CDR2 and CDR3 (CDR H2, CDR H3 or H2, H3) of the heavy chain variable domain. Typically, each heavy chain variable region has three CDRs (HCDR1, HCDR2, HCDR3) and each light chain variable region has three CDRs (LCDR1, LCDR2, LCDR3). The amino acid sequence boundaries of the CDRs can be determined by any one of a variety of known methods, including the "Kabat" numbering convention (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering convention (see Al-Lazikani et al., (1997) JMB 273:927-948), and the ImMunoGenTics (IMGT) numbering convention (see Lefranc MP, Immunologist, 7, 132-136 (1999); Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003)). For example, in a typical format, according to the Kabat rules, the CDR amino acid residues in the heavy chain variable region (VH) are numbered 31 to 35 (HCDR1), 50 to 65 (HCDR2), and 95 to 102 (HCDR3), and the CDR amino acid residues in the light chain variable region (VL) are numbered 24 to 34 (LCDR1), 50 to 56 (LCDR2), and 89 to 97 (LCDR3).According to the Chothia rules, the CDR amino acid numbers in VH are 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), and the amino acid residue numbers in VL are 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). According to the combined CDR definition of Kabat and Chothia, the CDRs are composed of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH, and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL. According to the IMGT rules, the numbers of the CDR amino acid residues in VH are approximately 26 to 35 (CDR1), 51 to 57 (CDR2) and 93 to 102 (CDR3), and the numbers of the CDR amino acid residues in VL are approximately 27 to 32 (CDR1), 50 to 52 (CDR2) and 89 to 97 (CDR3). According to the IMGT rules, the CDR regions of an antibody can be determined by the program IMGT / DomainGap Align.

[0149] The term "antibody framework" refers to a part of a variable domain VL or VH that serves as a support for the antigen binding loops (CDRs) of said variable domain. In effect, it is a variable domain without the CDRs.

[0150] "Binding to IL-4R" means capable of interacting with human IL-4R (or an epitope, fragment thereof). The term "antigen-binding site" as used herein refers to the three-dimensional spatial site recognized by an antibody or antigen-binding fragment thereof.

[0151] The term "epitope" or "antigenic determinant" refers to a site on an antigen that is specifically bound by an immunoglobulin or antibody. An epitope usually includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-consecutive amino acids in a unique spatial conformation (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996)).

[0152] The terms "specific binding," "selective binding," "selectively binds," and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen. Typically, an antibody binds to an antigen at a concentration of about 10 -7 Smaller than M, e.g., about 10 -8 M, 10 -9 M or 10 -10 It binds with an affinity (KD) less than or equal to M.

[0153] The term "nucleic acid molecule" refers to a DNA molecule or an RNA molecule. A nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA. A nucleic acid is "operatively linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operatively linked to a coding sequence if it affects the transcription of the coding sequence.

[0154] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid linked to it. In one embodiment, the vector is a "plasmid," which refers to a circular double stranded DNA circle to which other DNA segments can be ligated. In another embodiment, the vector is a viral vector to which other DNA segments can be ligated into the viral genome. The vectors disclosed herein can either autonomously replicate in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) or, after introduction into a host cell, can integrate into the genome of the host cell and thus replicate along with the host genome (e.g., non-episomal mammalian vectors).

[0155] Methods for producing and purifying antibodies and antigen-binding fragments well known in the art are described, for example, in chapters 5-8 and 15 of the Cold Spring Harbor Antibody Laboratory Techniques Manual. Antigen-binding fragments can be prepared by the same conventional methods. The antibodies or antigen-binding fragments described in the invention have one or more human FR regions added to the CDR regions of non-human origin by genetic engineering methods. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) homepage http: / / imgt.cines.fr by aligning the IMGT human antibody variable region germline gene database with the MOE software, or from Immunoglobulin magazine, 2001 ISBN 012441351.

[0156] The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacterial, microbial, plant or animal cells. Bacteria susceptible to transformation include members of the enterobacteriaceae, such as strains of Escherichia coli and Salmonella, Bacillaceae, such as Bacillus subtilis, Pneumococcus, Streptococcus and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese Hamster Ovary cell line) and NS0 cells.

[0157] The engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. In one of the more preferred prior art, a mammalian expression system will cause glycosylation of the antibody, especially at the highly conserved N-terminal site of the Fc region. Positive clones are expanded in serum-free medium in a bioreactor to produce the antibody. The culture medium from which the antibody is secreted can be purified by conventional techniques. For example, purification is performed on an A or G Sepharose FF column with a conditioned buffer. Non-specifically bound components are washed off. Furthermore, the bound antibody is eluted by a pH gradient method, and the antibody fragments are detected and collected by SDS-PAGE. The antibody can be filtered and concentrated by conventional methods. Soluble mixtures and multimers may be removed by conventional methods such as molecular sieves, ion exchange, etc. The resulting product must be immediately frozen, such as at -70°C, or lyophilized.

[0158] The "identity" of an amino acid sequence is the percentage of amino acid residues in a first sequence that are identical to those in a second sequence, with alignment of amino acid sequences and gaps, if necessary, so that the percentage of sequence identity is maximized, and without any conservative substitutions being considered as part of the sequence identity. To measure the percentage of amino acid sequence identity, alignment can be achieved by various methods within the skill of the art, for example, publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters to be applied to measure alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences to be compared.

[0159] The term "anti-IL-4R antibody" or "antibody that binds IL-4R" refers to an antibody that can bind to IL-4R with sufficient affinity, e.g., such that the antibody can be used as a therapeutic agent that targets IL-4R. The extent of binding of an anti-IL-4R antibody to unrelated, non-IL-4R proteins can be less than about 10% of the binding of the antibody to IL-4R as measured, e.g., by radioimmunoassay (RIA). In some embodiments, an antibody that binds to IL-4R has a dissociation constant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, or ≦0.1 nM.

[0160] The term "peptide" refers to a compound fragment between an amino acid and a protein, consisting of two or more amino acid molecules linked together by peptide bonds, and is a structural and functional fragment of a protein; for example, hormones, enzymes, etc. are essentially peptides.

[0161] The term "sugar" refers to a biopolymer consisting of the three elements C, H, and O, and can be divided into monosaccharides, disaccharides, polysaccharides, and the like.

[0162] The term "fluorescent probe" refers to a type of fluorescent molecule that has characteristic fluorescence in the ultraviolet-visible-near infrared region and whose fluorescence properties (excitation and emission wavelengths, intensity, lifetime, polarization, etc.) can be sensitively changed by changes in the properties of the environment in which it exists, such as polarity, refractive index, viscosity, etc., and can change one or more of its fluorescence properties through non-covalent interactions with nucleic acids (DNA or RNA), proteins, or other macromolecular structures, and can be used to study the properties and behavior of macromolecular substances.

[0163] The term "alkyl group" refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing from 1 to 20 carbon atoms, preferably an alkyl group containing from 1 to 12 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 6-methylhexyl, 7-methylhexyl, 8-methylhexyl, 9-methylhexyl, 10-methylhexyl, 11-methylhexyl, 12-methylhexyl, 13-methylhexyl, 14-methylhexyl, 15-methylhexyl, 16-methylhexyl, 17-methylhexyl, 18-methylhexyl, 19-methylhexyl, 20-methylhexyl, 21-methylhexyl, 22-methylhexyl, 23-methylhexyl, 24-methylhexyl, 25-methylhexyl, 26-methylhexyl, 27-methylhexyl, 28-methylhexyl, 29-methylhexyl, 30-methylhexyl, 31-methylhexyl, 32-methylhexyl, 33-methylhexyl, 34-methylhexyl, 35-methylhexyl, 36-methylhexyl, 37-methylhexyl, 38-methylhexyl, 39-methylhexyl, 40-methylhexyl, 41-methylhexyl, 42-methylhexyl, 43-methylhex Examples of such groups include 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched chain isomers thereof.More preferably, it is an alkyl group containing 1 to 6 carbon atoms, non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and the like. The alkyl group may be substituted or unsubstituted, and when substituted, the substituent may be substituted at any available attachment point, and is preferably one or more groups independently selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxy groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups, oxo groups, carboxy groups, or carboxylate groups.

[0164] The term "heteroalkyl group" refers to an alkyl group containing one or more heteroatoms selected from N, O or S, wherein the alkyl group is as defined above.

[0165] The term "cycloalkyl group" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring containing 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, and more preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like, and polycyclic cycloalkyl groups include spiro, fused, and bridged cycloalkyl groups. "Carbocycle" refers to a ring system in a cycloalkyl group.

[0166] The term "spirocycloalkyl group" refers to a polycyclic group having 5 to 20 members, in which the monocyclic rings share one carbon atom (called a spiro atom), and may contain one or more double bonds, but none of the rings has a completely conjugated π-electron system. It is preferably 6 to 14 members, more preferably 7 to 10 members. Depending on the number of spiro atoms shared between the rings, spirocycloalkyl groups are divided into monospirocycloalkyl groups, bisspirocycloalkyl groups, and polyspirocycloalkyl groups, and are preferably monospirocycloalkyl groups and bisspirocycloalkyl groups. More preferably, they are 4 / 4, 4 / 5, 4 / 6, 5 / 5, or 5 / 6 monospirocycloalkyl groups. "Spirocarbocycle" refers to a ring system in a spirocycloalkyl group. Non-limiting examples of spirocycloalkyl groups are: [ka] Includes.

[0167] The term "fused cycloalkyl group" refers to an all-carbon polycyclic group having 5 to 20 members, in which each ring in the system shares an adjacent pair of carbon atoms with another ring in the system, in which one or more rings may contain one or more double bonds, but none of the rings has a fully conjugated pi-electron system. It is preferably 6 to 14 members, more preferably 7 to 10 members. Depending on the number of rings that it comprises, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic fused cycloalkyl groups, preferably bicyclic or tricyclic, more preferably 5-membered / 5-membered or 5-membered / 6-membered bicyclic alkyl groups. "Fused carbocyclic ring" refers to a ring system in a fused cycloalkyl group. Non-limiting examples of fused cycloalkyl groups are: [ka] Includes.

[0168] The term "bridged cycloalkyl group" refers to an all-carbon polycyclic group having 5 to 20 members, in which any two rings share two carbon atoms that are not directly connected, and may contain one or more double bonds, but none of the rings has a completely conjugated π-electron system. It is preferably 6 to 14 members, more preferably 7 to 10 members. Depending on the number of rings that it comprises, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic bridged cycloalkyl groups, and is preferably bicyclic, tricyclic or tetracyclic, and more preferably bicyclic or tricyclic. Non-limiting examples of bridged cycloalkyl groups are: [ka] Includes.

[0169] The cycloalkyl ring may be fused to an aryl group, a heteroaryl group, or a heterocycloalkyl ring, in which the ring connected to the parent structure is a cycloalkyl group, non-limiting examples include indanyl, tetrahydronaphthyl, benzocycloheptanyl, etc. The cycloalkyl group may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxy, or carboxylate.

[0170] The term "heterocyclyl group" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing 3 to 20 ring atoms, in which one or more of the ring atoms is nitrogen, oxygen or S(O). m (wherein m is an integer of 0 to 2), but does not include the ring moiety of -OO-, -OS-, or -SS-, and the remaining ring atoms are carbon. Preferably, it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms, and more preferably contains 3 to 6 ring atoms. Non-limiting examples of monocyclic heterocyclyl groups include pyrrolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothienyl, dihydroimidazolyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like, and preferably piperidinyl and pyrrolidinyl. Polycyclic heterocyclyl groups include spiro, fused, and bridged heterocyclyl groups. "Heterocycle" refers to the ring system in the heterocyclyl group.

[0171] The term "spiroheterocyclyl group" refers to a 5- to 20-membered polycyclic heterocyclyl group in which the monocyclic rings share one atom (called a spiro atom) in which one or more of the ring atoms is nitrogen, oxygen, or S(O). m (wherein m is an integer from 0 to 2), and the remaining ring atoms are carbon. It may contain one or more double bonds, but none of the rings has a completely conjugated π-electron system. It is preferably 6 to 14-membered, more preferably 7 to 10-membered. Depending on the number of spiro atoms shared between the rings, spiroheterocyclyl groups are divided into monospiroheterocyclyl groups, bisspiroheterocyclyl groups, and polyspiroheterocyclyl groups, and are preferably monospiroheterocyclyl groups and bisspiroheterocyclyl groups. More preferably, they are 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 5-membered, or 5-membered / 6-membered monospiroheterocyclyl groups. "Spiroheterocycle" refers to a ring system in a spiroheterocyclyl group. Non-limiting examples of spiroheterocyclyl groups are: [ka] Includes.

[0172] The term "fused heterocyclyl group" refers to a polycyclic heterocyclyl group having 5 to 20 members in which each ring in the system shares an adjacent pair of atoms with another ring in the system, one or more of the rings may contain one or more double bonds, but none of the rings has a completely conjugated pi-electron system, and in which one or more of the ring atoms is nitrogen, oxygen, or S(O) m (wherein m is an integer of 0 to 2), and the remaining ring atoms are carbon. It is preferably 6 to 14-membered, more preferably 7 to 10-membered. Depending on the number of rings constituting the heterocyclyl group, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclyl groups, preferably bicyclic or tricyclic, more preferably 5-membered / 5-membered or 5-membered / 6-membered bicyclic fused heterocyclyl groups. "Fused heterocycle" refers to a ring system in a fused heterocyclyl group. Non-limiting examples of fused heterocyclyl groups are: [ka] Includes.

[0173] The term "bridged heterocyclyl group" refers to a 5- to 14-membered polycyclic heterocyclyl group in which any two rings share two atoms that are not directly connected, and which may contain one or more double bonds, but in which no ring has a completely conjugated pi-electron system, and in which one or more ring atoms are not nitrogen, oxygen, or S(O) m (wherein m is an integer of 0 to 2), and the remaining ring atoms are carbon. It is preferably 6 to 14-membered, more preferably 7 to 10-membered. Depending on the number of constituting rings, it can be classified as a bicyclic, tricyclic, tetracyclic or polycyclic bridged heterocyclyl group, preferably a bicyclic, tricyclic or tetracyclic group, more preferably a bicyclic or tricyclic group. Non-limiting examples of bridged heterocyclyl groups are: [ka] Includes.

[0174] The heterocyclyl ring may be fused to an aryl group, a heteroaryl group, or a cycloalkyl ring, where the ring connected to the parent structure is a heterocyclyl group, non-limiting examples of which are: [ka] etc.

[0175] The heterocyclyl group may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxy groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups, oxo groups, carboxy groups, and carboxylate groups.

[0176] The term "aryl group" refers to an all-carbon monocyclic or fused polycyclic (i.e., rings sharing adjacent pairs of carbon atoms) group having a conjugated pi-electron system, preferably 6-14 members, such as phenyl and naphthyl groups. The aryl ring may be fused to a heteroaryl group, a heterocyclyl group, or a cycloalkyl ring, in which the ring connected to the parent structure is an aryl ring. An "aromatic ring" refers to a ring system in an aryl group. Non-limiting examples of aryl groups are: [ka] Including, The aryl group may be substituted or unsubstituted. When the aryl group is substituted, the substituent is preferably one or more groups independently selected from an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an alkylthio group, an alkylamino group, a halogen, a mercapto group, a hydroxy group, a nitro group, a cyano group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, a heteroaryl group, a cycloalkoxy group, a heterocycloalkoxy group, a cycloalkylthio group, a heterocycloalkylthio group, a carboxy group, or a carboxylate group, and is preferably a phenyl group.

[0177] The term "fused ring aryl group" may be an unsaturated fused ring structure having aromaticity, which contains 8 to 14 ring atoms and is formed by linking two or more ring structures by sharing two adjacent atoms, and preferably has 8 to 12 ring atoms. For example, it includes fully unsaturated fused ring aryl groups such as naphthalene and phenanthrene, and further includes partially saturated fused ring aryl groups such as benzo 3-8 membered saturated monocyclic cycloalkyl group and benzo 3-8 membered partially saturated monocyclic cycloalkyl group. "Fused aromatic ring" refers to a ring system in the fused ring aryl group. Specific examples of fused ring aryl groups are, for example, 2,3-dihydro-1H-indenyl, 1H-indenyl, 1,2,3,4-tetrahydronaphthyl, 1,4-dihydronaphthyl, etc.

[0178] The term "heteroaryl group" refers to a heteroaromatic system containing 1-4 heteroatoms and 5-14 ring atoms, of which the heteroatoms are selected from oxygen, sulfur and nitrogen. The heteroaryl group is preferably 5-12 membered, such as imidazolyl, furanyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, pyrrolyl, tetrazolyl, pyridyl, pyrimidinyl, thiadiazolyl, pyrazinyl, etc., preferably imidazolyl, pyrazolyl, pyrimidinyl or thiazolyl, more preferably pyrazolyl or thiazolyl. The heteroaryl ring may be fused to an aryl, heterocyclyl or cycloalkyl ring, of which the ring connected to the parent structure is a heteroaryl ring. "Heteroaromatic ring" refers to a ring system in a heteroaryl group. Non-limiting examples of heteroaryl groups are: [ka] Includes.

[0179] The heteroaryl group may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently selected from an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an alkylthio group, an alkylamino group, a halogen, a mercapto group, a hydroxy group, a nitro group, a cyano group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, a heteroaryl group, a cycloalkoxy group, a heterocycloalkoxy group, a cycloalkylthio group, a heterocycloalkylthio group, a carboxy group, or a carboxylate group.

[0180] The term "fused heteroaryl group" refers to an unsaturated fused ring structure having aromaticity, which contains 5 to 14 ring atoms (including at least one heteroatom) and is formed by linking two or more ring structures by sharing two adjacent atoms, and at the same time, contains a carbon atom, a nitrogen atom, and a sulfur atom that can be substituted by oxo, and is preferably a "5- to 12-membered fused heteroaryl group", a "7- to 12-membered fused heteroaryl group", a "9- to 12-membered fused heteroaryl group", etc., and examples thereof include a benzofuranyl group. , benzisofuranyl, benzothienyl, indolyl, isoindolyl, benzoxazolyl, benzimidazolyl, indazolyl, benzotriazolyl, quinolyl, 2-quinolinone, 4-quinolinone, 1-isoquinolinone, isoquinolyl, acridinyl, phenanthridinyl, benzopyridazinyl, phthalazinyl, quinazolinyl, quinoxalinyl, phenazinyl, pteridinyl, purinyl, naphthyridinyl, phenazinyl, phenothiazinyl, etc. "Fused heteroaromatic ring" refers to a ring system in a fused heteroaryl group.

[0181] The fused heteroaryl group may be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more groups independently selected from an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an alkylthio group, an alkylamino group, a halogen atom, a mercapto group, a hydroxy group, a nitro group, a cyano group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, a heteroaryl group, a cycloalkoxy group, a heterocycloalkoxy group, a cycloalkylthio group, a heterocycloalkylthio group, a carboxy group, or a carboxylate group.

[0182] The term "alkylene group (-CH2-)" refers to the moiety remaining after removing two hydrogen atoms from an alkane molecule, and includes straight and branched chain subgroups of 1 to 20 carbon atoms. Non-limiting examples of alkylene groups containing 1 to 6 carbon atoms include methylene (-CH2-), ethylidene (e.g., -CH2CH2- or -CH(CH3)-), propylidene (e.g., -CH2CH2CH2- or -CH(CH2CH3)-), and butylidene (e.g., -CH2CH2CH2CH2-). An alkylene group may be substituted or unsubstituted, and when substituted, the substituent may be substituted at any available point of attachment, with one or more groups independently selected from halogen, deuterium, hydroxy, nitro, cyano, or amino being preferred. Similarly, "alkenylene group", "heteroarylene group", "arylene group", "heterocycloalkylene group", and the like are as defined above.

[0183] The term "alkenylene group" refers to a straight-chain alkenyl group having 2 to 8 carbon atoms, preferably 2 to 6 carbon atoms, and more preferably 2 to 4 carbon atoms, and having at least one double bond at any position, and includes, for example, a vinylidene group, an allylene, a propenylene group, a butenylene group, a phenylene group, a butadienylene group, a pentenylene group, a pentadienylene group, a hexenylene group, and a hexadienylene group.

[0184] The term "alkynylene group" refers to a straight-chain alkynylene group having 2 to 8 carbon atoms, preferably 2 to 6 carbon atoms, and more preferably 2 to 4 carbon atoms, and having at least one triple bond at any position, and includes, for example, an ethynylene group, a propynylene group, a butynylene group, a pentynylene group, a hexynylene group, etc.

[0185] The term "alkoxy group" refers to -O-(alkyl group) and -O-(unsubstituted cycloalkyl group), where alkyl group is defined above. Non-limiting examples of alkoxy groups include methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, and cyclohexyloxy. An alkoxy group may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxy, or carboxylate.

[0186] The term "alkylthio group" refers to -S-(alkyl group) and -S-(unsubstituted cycloalkyl group), where alkyl group is defined above. Non-limiting examples of alkylthio groups include methylthio group, ethylthio group, propylthio group, butylthio group, cyclopropylthio group, cyclobutylthio group, cyclopentylthio group, and cyclohexylthio group. The alkylthio group may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more groups substituted with one or more substituents independently selected from alkyl group, alkenyl group, alkynyl group, alkoxy group, alkylthio group, alkylamino group, halogen, mercapto group, hydroxy group, nitro group, cyano group, cycloalkyl group, heterocycloalkyl group, aryl group, heteroaryl group, cycloalkoxy group, heterocycloalkoxy group, cycloalkylthio group, and heterocycloalkylthio group.

[0187] The term "hydroxyalkyl group" refers to an alkyl group substituted with a hydroxy group, wherein the alkyl group is as defined above.

[0188] The term "haloalkyl group" refers to an alkyl group substituted with a halogen, wherein the alkyl group is as defined above.

[0189] The term "deuterated alkyl group" refers to an alkyl group substituted with a deuterium atom, wherein the alkyl group is as defined above.

[0190] The term "hydroxy" refers to an -OH group.

[0191] The term "oxo" refers to a =O group, e.g., a carbon atom and an oxygen atom are linked by a double bond where a ketone or aldehyde group is formed.

[0192] The term "thio" refers to the group =S. For example, a carbon atom and a sulfur atom are joined by a double bond to form thiocarbonyl -C(S)-.

[0193] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0194] The term "amino group" refers to -NH2.

[0195] The term "cyano" refers to -CN.

[0196] The term "nitro group" refers to --NO.sub.2.

[0197] The term "carboxy" refers to -C(O)OH.

[0198] The term "aldehyde group" refers to --CHO.

[0199] The term "carboxylic acid ester group" refers to a -C(O)O(alkyl group) or a -C(O)O(cycloalkyl group), where alkyl and cycloalkyl groups are as defined above.

[0200] The term "acyl halide" refers to a compound that contains the group -C(O)-halogen.

[0201] The term "sulfonyl group" refers to -S(O)(O)-.

[0202] The term "sulfinyl group" refers to -S(O)-.

[0203] The term "amino-protecting group" refers to a group that is applied to the protection of an amino group known in the art, and is described in the literature ("Protective Groups in Organic Synthesis", 5 Th The amino protecting group in "C . Ed. TW Greene & P. ​​G M Wuts" is referred to. Preferably, the amino protecting group is a (C 1-10 (C alkyl or aryl group) may be an acyl group; 1-6 Alkyl group or C 6-10 aryl group) may be a sulfonyl group, and (C 1-6 Alkoxy group or C 6-10 The alkyl group may be a substituted or unsubstituted alkyl group such as a trityl group (Tr), 2,4-dimethoxybenzyl (DMB), p-methoxybenzyl (PMB) or benzyl group (Bn).

[0204] "Optionally" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes cases where the event or circumstance occurs and cases where the event or circumstance does not occur. For example, "a heterocycloalkyl group that is optionally substituted with an alkyl group" means that the alkyl group may or may not be present, and the description includes cases where the heterocycloalkyl group is substituted with an alkyl group and cases where the heterocycloalkyl group is not substituted with an alkyl group.

[0205] The term "pharmaceutical composition" refers to a mixture of one or more compounds described herein or their physiologically / pharmaceutical acceptable salts or prodrugs and other chemical components, together with other components such as physiologically / pharmaceutical acceptable vectors and excipients. The pharmaceutical composition is intended to facilitate administration to a living body and contribute to the absorption of the active ingredients to further exert biological activity.

[0206] The term "drug vector" as used in the present disclosure refers to a system that changes the way a drug enters the human body and its distribution in the body, controls the release rate of the drug, and transports the drug to a target organ. The release and targeting system of the drug vector can reduce the degradation and loss of the drug, reduce side effects, and improve bioavailability. For example, the polymer surfactants that are used as vectors can self-assemble to form aggregates of various forms due to their unique amphiphilic structure, and preferred examples include micelles, microemulsions, gels, liquid crystals, vesicles, etc. These aggregates have the ability to encapsulate drug molecules and have good permeability to membranes, making them good drug vectors.

[0207] The term "excipient" refers to an additive other than the main drug in a pharmaceutical formulation, and may be called an additive. For example, any of the following may be called an excipient: binders, fillers, disintegrants, and lubricants in tablets, base parts in ointments and creams that are semi-solid preparations, preservatives, antioxidants, flavorings, fragrances, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, and coloring agents in liquid preparations.

[0208] The term "diluent" is also called a filler, and its main use is to increase the weight and volume of the tablet. The addition of a diluent not only ensures a certain volume size, but also reduces the dosage deviation of the main ingredient, improves the compression moldability of the drug, etc. If the tablet drug contains an oily ingredient, an absorbent needs to be added to absorb the oily substance in order to maintain a "dry" state and contribute to the preparation into a tablet.

[0209] The compounds in the present disclosure may contain one or more asymmetric centers, and therefore may form enantiomers, diastereomers, and may be defined by absolute stereochemistry as (R)- or (S)-, or as used for amino acids, as (D)- or (L)-, other stereoisomeric forms. The present disclosure includes all possible isomers and their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or may be prepared by conventional methods, such as chromatography and fractional crystallization. Conventional methods for preparing / separating individual enantiomers include chiral synthesis from appropriate optically pure precursors, or resolution of the racemates (or racemates of salts or derivatives), for example, by chiral high performance liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other geometrically asymmetric centers, it is meant that the compounds include E and Z geometric isomers, unless otherwise stated. All tautomeric forms are also meant to be included.

[0210] In the chemical structures of the compounds described in this disclosure, [ka] indicates that the configuration is not specified, i.e., if chiral isomers are present in the chemical structure, [ka] The bond [ka] or [ka] In the chemical structure of the compound described in the present disclosure, [ka] The bond "Z" is not specified, i.e., it may be in the Z or E configuration, or it may include the two configurations simultaneously.

[0211] "Stereoisomer" refers to a compound composed of the same atoms bonded by the same bonds but with different, incompatible three-dimensional structures. In this disclosure, various stereoisomers and mixtures thereof are contemplated, and include "enantiomers," which refers to two stereoisomers whose molecules are non-superimposable mirror images of one another.

[0212] "Tautomer" refers to a proton migrating from one atom of a molecule to another atom of the same molecule. The present disclosure includes any tautomers of the compounds.

[0213] Any isotopically labeled derivatives of the compounds described in this disclosure or their medicamentable salts, or isomers thereof, are covered by this disclosure. Isotopically labelable atoms include, but are not limited to, hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, iodine, etc., each of which is an isotope. 2 H(D), 3 H, 11 C. 13 C. 14 C. 15 N, 18 F, 31 P, 32 P, 35 S, 36 Cl and 125 I, etc. Unless otherwise specified, when a position is specifically designated as deuterium (D), it is to be understood that the position is a deuterium having an abundance at least 3000 times greater than the natural abundance of deuterium, which is 0.015%, (i.e., at least 45% deuterium incorporated). [Brief description of the drawings]

[0214] [Figure 1] 1 shows a dose-response curve of the whole cell binding activity of a test protein against human IL-4Rα expressing cells. [Diagram 2]1 shows a dose-response curve of the blocking effect of the test protein on the IL-4 / IL-13 signaling pathway. [Diagram 3] 1 shows the endocytosis rate-time curve of the test protein in TF-1 cells. [Figure 4] 4 shows time-concentration curves of ADC-2 and free Budesonide in each tissue after intratracheal administration. [Diagram 5] Counts of white blood cells and differential cells in bronchoalveolar lavage fluid in Test Example 6. [Figure 6] Counts of white blood cells and differential cells in bronchoalveolar lavage fluid in Test Example 7. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0215] The preparation of the compounds and pharma- ceutical salts described in this disclosure are further illustrated in the following examples, which are not intended to limit the scope of this disclosure.

[0216] Experimental methods for which specific conditions are not specified in the examples of this disclosure generally follow conventional conditions or conditions recommended by the raw material or product manufacturers. Reagents for which specific sources are not specified are conventional reagents that are commercially available.

[0217] The NMR shift (δ) is 10 -6 The units are shown in ppm. A Bruker AVANCE-400 nuclear magnetic resonance spectrometer was used for the NMR measurements, and the measurement solvent was deuterated dimethyl sulfoxide (DMSO-d 6 ), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD), and the internal standard was tetramethylsilane (TMS).

[0218] For MS measurements, a Shimadzu 2010 Mass Spectrometer or an Agilent 6110A MSD mass spectrometer was used.

[0219] For HPLC measurements, Shimadzu LC-20A systems, Shimadzu LC-2010HT series or Agilent 1200 LC high performance liquid chromatograph (Ultimate XB-C18 3.0×150 mm column or Xtimate C18 2.1×30 mm column) were used.

[0220] For chiral HPLC analysis and measurements, Chiralpak IC-3 100×4.6mm ID, 3μm, Chiralpak AD-3 150×4.6mm ID, 3μm, Chiralpak AD-3 50×4.6mm ID, 3μm, Chiralpak AS-3 150×4.6mm ID, 3μm, Chiralpak AS-3 100×4.6mm ID, 3μm, ChiralCel OD-3 150×4.6mm ID, 3μm, Chiralcel OD-3 100×4.6mm ID, 3μm, ChiralCel OJ-H 150×4.6mm ID, 5μm, Chiralcel OJ-3 150×4.6mm A column with ID of 3 μm was used, and Yantai Yellow Sea HSGF254 or Qingdao GF254 silica gel plates were used as silica gel plates for thin layer chromatography. The specifications for the silica gel plates used in thin layer chromatography (TLC) were 0.15-0.2 mm, and the specifications for separation and purification of products by thin layer chromatography were 0.4-0.5 mm.

[0221] For column chromatography, Yantai Huanghai silica gel 100-200 mesh, 200-300 mesh or 300-400 mesh silica gel was generally used as the vector.

[0222] The chiral preparative columns used were DAICEL CHIRALPAK IC (250×30 mm, 10 μm) or Phenomenex-Amylose-1 (250×30 mm, 5 μm).

[0223] Combiflash Rf150 (TELEDYNE ISCO) was used as the CombiFlash high-speed preparative chromatograph.

[0224] Kinase mean inhibition rate and IC 50 The values ​​were measured using a plate reader NovoStar (BMG, Germany).

[0225] Known starting materials according to the present disclosure may be synthesized by or according to methods known in the art, or may be purchased from companies such as ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Shaoyuan Chemical Technology (Accela ChemBio Inc.), and Darui Chemical.

[0226] In the examples, unless otherwise stated, all reactions can be carried out in an argon or nitrogen atmosphere.

[0227] An argon or nitrogen atmosphere refers to an argon or nitrogen balloon of approximately 1 L volume connected to the reaction flask.

[0228] Hydrogen atmosphere refers to a hydrogen balloon of approximately 1 L volume connected to the reaction flask.

[0229] For the pressurized hydrogenation reaction, a Parr 3916EKX hydrogenation apparatus and a Seiran QL-500 hydrogen generator or an HC2-SS hydrogenation apparatus were used.

[0230] The hydrogenation reaction was generally carried out by repeating the process of evacuating and refilling with hydrogen three times.

[0231] A CEM Discover-S 908860 microwave reactor was used for microwave reactions.

[0232] In the examples, unless otherwise specified, the solution refers to an aqueous solution.

[0233] In the examples, unless otherwise specified, the reaction temperature is room temperature, 20 to 30°C.

[0234] In the examples, thin layer chromatography (TLC) was used to monitor the progress of the reaction. The developing solvents used in the reaction, the eluent system of column chromatography for purifying the compounds, and the developing solvent system of thin layer chromatography included A: dichloromethane / methanol system, B: n-hexane / ethyl acetate system, C: petroleum ether / ethyl acetate system, and D: petroleum ether / ethyl acetate / methanol, and the volume ratio of the solvents was adjusted according to the polarity of the compounds, and may be adjusted by adding a small amount of basic or acidic reagent such as triethylamine and acetic acid.

[0235] Preparation of 1.0M Tris buffer pH=8.30±0.1: 6.0 g of tris was weighed into a 50 mL measuring flask, 40 mL of purified water was added, and the mixture was shaken to dissolve. 1.2 mL of concentrated hydrochloric acid was added dropwise to adjust the pH to 8.30, and the volume was then adjusted to constant with purified water.

[0236] Preparation of Buffer A: In a 2.0 L container, KH2PO4 (8.50 g), K2HPO4 (8.56 g), NaCl (5.86 g) and EDTA (1.50 g) were added, 1.6 L of water for injection was added, and the mixture was stirred for 0.5 hours until completely dissolved. The volume was then adjusted to 2.0 L with water for injection, and the pH was measured to be 6.30±0.1.

[0237] The abbreviations used in the following experiments have the following meanings: DAST: diethylaminosulfur trifluoride, THF: tetrahydrofuran, NMP: N-methylpyrrolidone, DCM: dichloromethane, m-CPBA: metachloroperbenzoic acid, DIEA: N,N-diisopropylethylamine, TEA: triethylamine, Boc: tert-butoxycarbonyl group, MeOH: methanol, Et2O: diethyl ether.

[0238] The preparation and purification methods of the 25G7 and 7B10 antibodies in this application are described in a patent document having application number WO2020038454A1, the contents of which are incorporated in their entirety into this disclosure.

[0239] Example 1: Preparation of N-((10S)-10-benzyl-1-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-1,6,9,12,15-pentaoxy-3-oxo-5,8,11,14-tetrapolyethyleneglycol-16-yl)-1-(2-bromoacetamido)-3,6,9,12-tetraoxopentadecane-15-amide (Compound 1) [ka] [ka]

[0240] Step 1) Preparation of (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanine tert-butyl ester (compound 1b) Compound 1a (5.14 g, 20.0 mmol, 1.0 eq) and L-phenylalanine tert-butyl hydrochloride (7.08 g, 20.0 mmol, 1.0 eq) were placed in a 250 mL three-neck flask and clarified with anhydrous DMF (60 mL). The mixture was cooled in an ice bath, and HATU (9.12 g, 24.0 mmol, 1.2 eq) and DIEA (7.74 g, 60.0 mmol, 3.0 eq) were added, and the mixture was reacted for 2 hours in an ice bath. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated saline, dried, and concentrated under reduced pressure to obtain compound 1b (11.5 g, 100% yield), and the crude product can be directly put into the next step. MS(ESI): m / z 580.3[M+Na] +. 1 H NMR(400 MHz,DMSO-d6)δ 8.21-8.16(m,1H),8.12-8.05(m,1H),7.91-7.87(m,2H),7.72-7.68(m,2H),7.64-7.59(m,1H),7.44-7.38(m,2H),7.36-7.25(m ,4H),7.23-7.18(m,3H),4.41-4.18(m,4H),3.76-3.72(m,2H),3.68-3.61(m,2H),2.98-2.89(m,2H),2.69(s,2H),1.30(s,9H).

[0241] Step 2) Preparation of tert-butyl glycyl-L-phenylalanine (Compound 1c) Compound 1b (5.57 g, 10.0 mmol, 1.0 eq) was placed in a 250 mL three-neck flask, clarified with anhydrous DCM (60 mL), and cooled in an ice bath. Piperidine (8.5 g, 100.0 mmol, 10.0 eq) was slowly added, and after addition was complete, the reaction was allowed to proceed with stirring at room temperature for about 2 hours. The reaction solution was directly concentrated to dryness, and the crude product was purified by column chromatography to obtain compound 1c (2.70 g, 81% yield). MS(ESI): m / z 358.3[M+H] + .

[0242] Step 3) Preparation of (1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16-pentoxy-4-tetrapolyethylene glycol-19-oxy)glycylglycyl-L-phenylalanine tert-butyl (Compound 1d) In a 100mL three-neck flask, raw materials 1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16-pentaoxy-4-tetrapolyethylene glycol-19-oleic acid (1.0g, 2.05mmol, 1.0eq, from Tonglai Chemical) and 1c (0.69g, 2.05mmol, 1.0eq) were added, and the mixture was clarified by adding anhydrous DMF (25mL) and cooled in an ice bath. HATU (1.01g, 2.67mmol, 1.3eq) and DIEA (529mg, 4.10mmol, 2.0eq) were added, and the mixture was reacted for 1 hour in an ice bath. Water was added to the reaction solution, which was then extracted with ethyl acetate, washed with saturated saline, dried, and concentrated under reduced pressure to obtain compound 1d (1.60g, 97% yield). MS(ESI): m / z 805.3[M+H] + .

[0243] Step 4) Preparation of (1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16-pentaoxy-4-tetrapolyethylene glycol-19-oxy)glycylglycyl-L-phenylalanine (compound 1e) Compound 1d (1.60 g, 1.99 mmol, 1.0 eq) was added to a 100 mL one-neck flask, and anhydrous dichloromethane (16 mL) was added to dissolve. Trifluoroacetic acid (8 mL) was added at room temperature, and the reaction was carried out while stirring for 2 hours. The reaction solution was directly concentrated to dryness, dissolved by adding ethyl acetate, washed with saturated saline, dried, and concentrated under reduced pressure to obtain compound 1e (1.23 g, 83% yield). MS(ESI): m / z 749.3[M+H] + .

[0244] Step 5) Preparation of (9H-fluoren-9-yl)methyl (2-(((2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethoxy)methyl)amino)-2-oxoethyl)carbamate (compound 1f) The raw materials budesonide (1.29 g, 3.0 mmol, 1.0 eq), (2-((((9H-fluoren-9-yl) methoxy) carbonyl) amino) acetamido) methyl acetate (1.16 g, 3.15 mmol, 1.0 eq, prepared according to the method of reference "Tetrahedron, 2018, 74 (15), 1951-1956") and p-toluenesulfonic acid pyridine salt (75 mg, 0.30 mmol, 0.1 eq) were added to a 100 mL three-neck flask. Anhydrous tetrahydrofuran (20 mL) was added at room temperature and the mixture was refluxed for 4 hours to react. The reaction solution was directly concentrated to dryness, and the crude product was purified by column chromatography to obtain 1f (0.54 g, 24% yield). MS(ESI): m / z 739.4[M+H] + . 1 H NMR(400 MHz,DMSO-d6)δ 8.76-8.69(m,1H),7.91-7.84(m,2H),7.73-7.67(m,2H),7.63-7.57(m,1H),7.43-7.58(m,2H),7.35-7.23(m,3H),6.13(d,J=7.5 Hz,1H),5.91(s,2H),5.15-4.97(m,1H),4.73-4.45(m,5H),4.30-4.09(m,5H),3.65-3. 59(m,2H),2.29-2.21(m,1H),2.11-1.87(m,2H),1.74-1.21(m,11H),1.09-0.77(m,8H).

[0245] Step 6) Preparation of 2-amino-N-((2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethoxy)methyl)acetamide (compound 1g) Compound 1f (540 mg, 0.73 mmol, 1.0 eq) was placed in a 25 mL one-neck flask and dissolved in anhydrous dichloromethane (10 mL). The mixture was cooled in an ice bath, DBU (167 mg, 1.10 mmol, 1.5 eq) was added, and the mixture was reacted for 30 minutes with stirring. Water was added to separate the mixture, and the aqueous phase was extracted twice with dichloromethane, washed with saturated saline, and concentrated under reduced pressure to obtain compound 1g (450 mg, 100% yield). The crude product can be used directly in the next reaction. MS(ESI): m / z 517.4[M+H] + .

[0246] Step 7) (9H-fluoren-9-yl)methyl((10S)-10-benzyl-1-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-do Preparation of decahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-1,6,9,12,15,18-hexaoxo-3,21,24,27,30-pentaoxo-5,8,11,14,17-pentaaminotriacontan-32-yl)carbamate (compound 1h) Compound 1g (0.45 g crude product, converted to 0.73 mmol, 1.0 eq) and 1e (0.55 g, 0.73 mmol, 1.0 eq) were placed in a 100 mL three-neck flask, and anhydrous DMF (9 mL) was added to clarify. The mixture was cooled in an ice bath, and HATU (361 mg, 0.95 mmol, 1.3 eq) and DIEA (189 mg, 1.46 mmol, 2.0 eq) were added in sequence, and the mixture was reacted with stirring for 1 hour. Water was added to the reaction solution, and the mixture was extracted twice with dichloromethane, and the organic phases were combined, washed once with saturated saline, dried, and concentrated under reduced pressure to obtain a crude product. The crude product was slurried and purified in a mixed solvent of petroleum ether and ethyl acetate to obtain compound 1h (710 mg, 78% yield). MS(ESI): m / z 1269.7[M+Na] + .

[0247] Step 8) Preparation of 1-amino-N-((10S)-10-benzyl-1-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-1,6,9,12,15-pentaoxo-3-oxo-5,8,11,14-polytetraethyleneglycol-16-yl-3,6,9,12-tetraoxopentadecane-15-amide (compound 1i) Compound 1h (350 mg, 0.28 mmol, 1.0 eq) was placed in a 25 mL one-neck flask and clarified by adding anhydrous dichloromethane (10 mL). The mixture was cooled in an ice bath, DBU (64 mg, 0.42 mmol, 1.5 eq) was added, and the mixture was reacted with stirring for 1 hour. The reaction solution was directly concentrated to dryness to obtain a crude product, which was further purified by column chromatography to obtain compound 1i (289 mg, 89% yield). MS(ESI): m / z 1025.6[M+H] + .

[0248] Step 9) Preparation of N-((10S)-10-benzyl-1-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-1,6,9,12,15-pentaoxy-3-oxo-5,8,11,14-tetrapolyethyleneglycol-16-yl)-1-(2-bromoacetamido)-3,6,9,12-tetraoxopentadecane-15-amide (compound 1) Bromoacetic acid solid (35.2 mg, 0.127 mmol, 1.0 eq) and EEDQ (63 mg, 0.25 mmol, 2.0 eq) were placed in a 25 mL Schlenk-Tube, and anhydrous DMF (5 mL) was added to clarify. The mixture was left at room temperature and reacted for 1 hour with stirring. Compound 1i (130 mg, 0.127 mmol, 1.0 eq) was dissolved in DMF (2 mL), and this solution was added dropwise to the activated ester solution prepared above. After addition was complete, the mixture was left at room temperature and reacted for 2 hours. Water was added to the reaction system, and the mixture was extracted twice with ethyl acetate. The organic phases were combined, washed with saturated saline, dried, and concentrated under reduced pressure to obtain a crude product, which was further purified by column chromatography to obtain compound 1 (39 mg, 27% yield). MS(ESI):m / z 1145.5 / 1147.6[M+1] + . 1H NMR(400 MHz,CDCl3)δ 8.62-8.57(m,1H),8.34-8.28(m,2H),8.19-8.11(m,2H),8.03-7.99(m,1H),7.30-7.17(m,6H),6.15(d,J=9.6 Hz,1H),5.92(s,2H),5.18-5.03(m,1H),4.72-4.47(m,5H),4.29-4.15(m,2H),3.78-3.42(m,27H),3.27-3. 21(m,2H),3.06-2.84(m,2H),2.41-2.28(m,3H),2.06-1.73(m,2H),1.60-1.24(m,10H),1.01-0.82(m,8H).

[0249] Example 2: N-((10S)-10-benzyl-1-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5] Indeno[1,2-d][1,3]dioxol-8b-yl)-1,6,9,12,15-pentoxy-3-oxo-5,8,11,14-tetrapolyethylene glycol-16-yl)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12-tetraoxopentadecane-15-amide [ka] [ka]

[0250] Step 1) Preparation of (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanine (compound 2a) Compound 1b (1.50 g, 2.69 mmol, 1.0 eq) was placed in a 100 mL one-neck flask and dissolved in 4 M HCl in 1,4-dioxane (20 mL). The mixture was stirred at room temperature for 4 hours until the raw material 1b was completely reacted. The reaction solution was directly concentrated to dryness to obtain compound 1a (1.34 g, 100% yield). MS(ESI): m / z 502.3[M+1] +

[0251] Step 2) Preparation of (9H-fluoren-9-yl)methyl-((10S)-10-benzyl-1-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-1,6,9,12,15-pentaoxo-3-oxy-5,8,11,14-tetrapolyethylene glycol-16-yl)carbamate (compound 2b) Compound 2a (0.97 g, 1.63 mmol, 1.0 eq) and 1g (1.0 g, 1.95 mmol, 1.2 eq) were placed in a 100 mL three-neck flask. DMF (20 mL) was added and cooled in an ice bath. HATU (927 mg, 2.44 mmol, 1.5 eq) and DIEA (420 mg, 3.26 mmol, 2.0 eq) were added in order, and after addition was completed, the reaction was carried out for 1 to 2 hours in an ice bath. The reaction solution was concentrated and purified by column chromatography to obtain compound 2b (860 mg, 53% yield). MS(ESI): m / z 1000.5[M+1] +

[0252] Step 3 Preparation of (2S)-2-(2-(2-aminoacetamido)acetamido-N-(2-(((2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethoxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide (compound 2c) Compound 2b (860 mg, 0.86 mmol, 1.0 eq) was placed in a 25 mL one-neck flask and clarified by adding anhydrous DMF (10 mL). The mixture was cooled in an ice bath, and DBU (196 mg, 1.29 mmol, 1.5 eq) was added and reacted with stirring for 30 minutes. The reaction solution was directly concentrated and purified by column chromatography to obtain compound 2c (485 mg, 72% yield). MS(ESI): m / z 778.4[M+1] +

[0253] Step 4) N-((10S)-10-benzyl-1-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5]ine Preparation of deno[1,2-d][1,3]dioxol-8b-yl)-1,6,9,12,15-pentoxy-3-oxo-5,8,11,14-tetrapolyethylene glycol-16-yl)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12-tetraoxopentadecane-15-amide (compound 2d) Compound 2c (480 mg, 0.62 mmol, 1.0 eq) and 1-(2,5-dioxy-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12-tetraoxoglutaric acid-15-oleic acid (213 mg, 0.62 mmol, 1.0 eq, from Tong Lai Chemical) were placed in a 100 mL three-neck flask, and anhydrous DMF (10 mL) was added to clarify the mixture. The mixture was cooled in an ice bath, and HATU (352 mg, 0.93 mmol, 1.5 eq) and DIEA (159 mg, 1.23 mmol, 2.0 eq) were added in that order, and the mixture was reacted with stirring for 1 hour. The reaction solution was directly concentrated and purified by column chromatography to obtain compound 2d (360 mg, 53% yield). MS(ESI): m / z 1105.6[M+1] + 1 H NMR(400 MHz,DMSO-d6)δ 8.61-8.56(m,1H),8.32-8.26(m,1H),8.22-8.13(m,2H),8.03-7.96(m,1H),7.39-7 .20(m,6H),7.02(s,2H),6.17-6.13(m,1H),5.92(s,2H),5.16-4.99(m,1H),4.76-4 .40(m,6H),4.29-4.16(m,2H),3.64-3.19(m,26H),3.09-3.02(m,1H),2.86-2.79(m ,1H),2.41-2.26(m,3H),2.12-1.90(m,2H),1.78-1.25(m,10H),1.03-0.83(m,8H).

[0254] Example 3: Preparation process of 25G7-Fab protein The antibody gene sequence was synthesized and subcloned into the pcDNA3.4 vector. The ligation product was transformed into Top10 competent cells, and positive clones were selected and expanded. The clones were inoculated into culture medium and expanded, and a large amount of plasmid containing the deoxyribonucleic acid sequence of the 25G7-Fab antibody was extracted. The expression plasmid and transfection reagent were mixed by lipofection, then added to Expi293 cells, and cultured in a constant temperature incubator for 5 days to obtain a cell culture. The cell culture was centrifuged, the supernatant was taken, and the clarified liquid was collected by filtration to remove cell debris. The target protein was captured by a kappa select chromatography column. The target protein was filtered, and the protein concentration was obtained by measuring the A280 absorbance value with Nanodrop and dividing it by the extinction coefficient, and the final yield was determined by multiplying the volume. The protein was subjected to gel electrophoresis, size exclusion chromatography, SDS-PAGE, SEC-HPLC and endotoxin detection to obtain the final product, 25G7-Fab.

[0255] Example 4: Preparation process of AZD1402-TAG protein AZD1402-TAG (the protein sequence is derived from SEQ ID NO: 1 in patent WO2020200960A1, and a His tag was fused to the C-terminus of the sequence to facilitate protein preparation and purification, and was finally named AZD1402-TAG).

[0256] AZD1402-TAG TIFF2025508665000080.tif37155

[0257] The coding gene sequences of the above proteins were synthesized and subcloned into pcDNA3.4. The expression plasmid and transfection reagent were mixed and incubated at 37°C for 15 minutes, and the mixture was added dropwise to HEK293 cell solution. The cell solution was cultured on a shaker at 37°C for one week, and the cell culture was centrifuged and the supernatant was taken. After equilibrating the nickel affinity column chromatography column with an imidazole-free buffer, the protein sample was run through the nickel affinity column chromatography column, and then equilibrated again with an imidazole-free buffer, and the column-bound protein was eluted with a buffer containing a high concentration of imidazole. The eluted protein was then transferred to a dialysis bag and dialyzed in 1xPBS, which was replaced with PBS storage buffer. Upon detection, the target protein was obtained.

[0258] Example 5: Preparation of antibody drug conjugate ADC-1 [ka]

[0259] Preparation of Buffer A: To a 2.0 L container, add KH2PO4 (8.50 g), K2HPO4 (8.56 g), NaCl (5.86 g) and EDTA (1.50 g), add 1.6 L of water for injection, and stir for 0.5 hours until completely dissolved. Then, adjust the volume to 2.0 L with water for injection. The pH was measured to be 6.30±0.1.

[0260] At 37°C, the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (2.5 mM, 100.0 μL, 0.250 mmol) was added to an aqueous solution of Buffer A (0.05 M aqueous solution of buffer with pH=6.3, 10.0 mg / mL, 0.5 mL, 0.125 mmol) of antibody 25G7-Fab (heavy chain sequence shown in SEQ ID NO:50, light chain sequence shown in SEQ ID NO:45), placed in a water bath shaker, and reacted with shaking at 37°C for 3 hours to terminate the reaction. The reaction solution was cooled to 25°C in a water bath.

[0261] 1.0M Tris buffer (50μL) was added to the reaction solution, and compound 1 (1.43mg, 1.250mmol) was dissolved in 25μL of DMSO and added to the reaction solution, placed in a water bath shaker, and reacted with shaking at 25℃ for 3 hours to stop the reaction. The reaction solution was desalted and purified with a Sephadex G25 gel column (elution phase: buffer A) and concentrated with an ultrafiltration tube to obtain the title product ADC-1 in buffer A buffer (6.06mg / mL, 0.60mL), which was then frozen and stored at 4℃.

[0262] Example 6: Preparation of antibody drug conjugate ADC-2 [ka]

[0263] At 37°C, the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10.0 mM, 1.50 mL, 15.0 mmol) was added to an aqueous solution of antibody 25G7-Fab in buffer A (0.05 M aqueous buffer with pH=6.3, 10.0 mg / mL, 6.0 mL, 1.50 mmol), and the mixture was placed in a water bath shaker and reacted with shaking at 37°C for 3 hours to terminate the reaction. The reaction solution was cooled to 25°C in a water bath.

[0264] Compound 2 (12.2 mg, 12.0 mmol) was dissolved in 300 μL of DMSO, added to the above reaction solution, placed in a water bath shaker, and reacted with shaking at 25 ° C for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified with a Sephadex G25 gel column (elution phase: buffer A) and concentrated with an ultrafiltration tube to obtain the title product ADC-2 in buffer A buffer (3.65 mg / mL, 12.8 mL), which was frozen and stored at 4 ° C.

[0265] Biological evaluation The present disclosure will be further described below in conjunction with test examples, but these examples do not limit the scope of the present disclosure.

[0266] Test Example 1: Detection of affinity of antibody-drug conjugate ADC-2 to human IL-4Rα protein Test Protein Antibody-drug conjugate ADC-2, 25G7-Fab, AZD1402-TAG

[0267] Experimental Method The affinity of antibody-drug conjugates ADC-2, 25G7-Fab and AZD1402-TAG to human IL-4Rα antigen protein was detected by surface plasmon resonance (SPR). An SA sensor chip (Cytiva) was placed in a Biacore 8K (Cytiva), and biotin-labeled human IL-4Rα protein (SinoBiological, CAT#10402-H08H-B) was captured on the SA sensor chip. HBS-EP+ buffer solution (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) was used as the mobile phase. Each test protein was serially diluted 2-fold with HBS-EP+ buffer solution and then injected as an analyte. The flow rate was 30 μL / min, and the detection time for ADC-2, 25G7-Fab and AZD1402-TAG was set to 100 seconds for binding and 600 seconds for dissociation. Analysis was performed using Biacore 8K evaluation software (Cytiva) to obtain affinity data for each test protein.

[0268] Experimental Results [Table 3]

[0269] As shown in Table 1, the KD value of ADC-2 binding to human IL-4Rα antigen protein was 11.0 pM, which is slightly better than those of AZD1402-TAG and 25G7-Fab.

[0270] Test Example 2: Total cell binding activity of antibody-drug conjugate ADC-2 to human IL-4Rα expressing cells 1. Test protein The antibody drug conjugates are ADC-2, 25G7-Fab, hu25G7 (heavy chain sequence shown in SEQ ID NO: 44 and light chain sequence shown in SEQ ID NO: 45), and AZD1402-TAG.

[0271] 2. Experimental method TF-1 and Karpas299 cell lines were identified as capable of expressing IL-4Rα. TF-1 and Karpas299 cells in logarithmic growth phase were harvested, counted, and adjusted to a cell density of 1 × 10 5 Cells were seeded in a round-bottom 96-well plate at 100 cells / well. The cells were incubated with various concentrations of anti-IL-4R antibodies (25G7, naked antibody 25G7-Fab), antibody-drug conjugates ADC-2 and AZD1402-TAG for 45 min at 4°C, and the antibodies were washed off with FACS Buffer (PBS containing 2% FBS), followed by staining with fluorescently labeled secondary antibodies. For 25G7-Fab, hu25G7 and ADC-2, a FITC-coupled anti-human IgG (Fab specific) secondary antibody (Sigma, CAT#F5512-1ML) was used, and for AZD1402-TAG, a FITC-coupled anti-His antibody (GenScript, CAT#A01620) was used. The cells were incubated with the secondary antibodies for 30 min at 4°C, washed twice with FACS Buffer, and detected by a flow cytometer (BD, FACS Celesta), and the detection results were analyzed with FlowJo (FlowJo, LLC) software.

[0272] 3. Experimental results [Table 4]

[0273] The results are shown in Figure 1 and Table 2. EC binding of ADC-2 to TF-1 cells 50 The value was 0.107 nM, which is comparable to the naked antibodies 25G7-Fab and hu25G7. AZD1402-TAG has strong binding activity to TF-1 cells, but the data cannot be compared with ADC-2, 25G7-Fab, etc., because the detection antibody is different.

[0274] Test Example 3: Blocking effect of antibody-drug conjugate ADC-2 on IL-4 / IL-13 signaling pathway 1. Test protein Antibody-drug conjugate ADC-2, 25G7-Fab.

[0275] 2. Experimental method STAT6 activation is a key step in activating the IL-4 / IL-13 signaling pathway. In this example, the blocking effect of ADC-2 on the IL-4 / IL-13 signaling pathway was confirmed by HEK-Blue TM The IL-4 / IL-13 signal transduction pathway was evaluated using an IL-4 / IL-13 reporter gene cell line. The cells were purchased from Invivogen (Cat#hkb-il413), and the human STAT6 gene and the secreted alkaline phosphatase reporter gene (SEAP), which was inducibly expressed by phosphorylated STAT6, were overexpressed in the cells. The activation level of the IL-4 / IL-13 signal transduction pathway was evaluated by detecting the amount of SEAP secreted into the cell culture supernatant using QUANTI-Blue, a SEAP substrate. HEK-Blue cells in logarithmic growth phase TM After harvesting, IL-4 / IL-13 cells were cultured at a density of 5 × 10 5 The cells were adjusted to 100 μL / well and added to a 96-well flat-bottom culture plate, and incubated for 24 hours under conditions of 37 ° C and 5% CO2. Then, 20 μL of serially diluted test protein and 20 μL of recombinant human IL-4 or IL-13 were added to each well, and the cell culture plate was placed in an incubator at 37 ° C and 5% CO2 for 20 to 24 hours. After incubation, the cell culture plate was removed, 20 μL of cell culture supernatant was aspirated from each well and transferred to another 96-well plate, and 180 μL of QUANTI-Blue coloring solution preheated at 37 ° C was added to each well, and the light absorption value at a wavelength of 620 nm was detected after incubation at 37 ° C for 1 hour.

[0276] 3. Experimental results [Table 5]

[0277] As shown in Figure 2 and Table 3, each test protein had a blocking effect on STAT6 activation induced by recombinant human IL-4 and IL-13, and the IC10 of ADC-2 blocking the IL-4 signaling pathway was significantly higher than that of ADC-2. 50 value of 0.68 nM and IC of 25G7-Fab 50 The IC value of ADC-2 for blocking the IL-13 signaling pathway was close to 0.61 nM. 50 value of 10.60 nM and IC of 25G7-Fab 50 This corresponds to a value of 14.59 nM.

[0278] Test Example 4: Endocytosis activity of antibody-drug conjugate ADC-2 in TF-1 cells 1. Test protein Antibody-drug conjugates ADC-2, 25G7-Fab, hu25G7, and AZD1402-TAG.

[0279] 2. Experimental method TF-1 cells were harvested from well-growing cells and then cultured at a density of 1 × 10 6The cells were adjusted to cells / mL, added to a 96-well cell culture plate at 100 μL / well and placed at 4 ° C. Each test protein was added to a final concentration of 2 nM, incubated at 4 ° C for 1 hour, and then placed in a cell incubator to continue culturing under conditions of 4 ° C and 5% CO2. One culture plate was placed at each time point, during which the culture plates were removed at different incubation times, washed with FACS buffer, centrifuged, and further incubated with fluorescently labeled secondary antibodies. 25G7-Fab, hu25G7, and ADC-2 were labeled with anti-human IgG (Fab-specific) secondary antibodies coupled with FITC, and AZD1402-TAG was labeled with anti-His antibodies coupled with FITC. The cells and secondary antibodies were incubated at 4 ° C for 30 minutes, then washed twice with FACS Buffer, and detected by a flow cytometer (BD, FACS Celesta), and the detection results were analyzed with FlowJo (FlowJo, LLC) software. The calculation formula for the endocytosis rate at a specific time point is as follows: (gMFI Test Ab at time X -gMFI ISO Ab at time X ) / (gMFI Test Ab at time zero -gMFI ISO Ab at time zero )×100%

[0280] 3. Experimental results The experimental results are shown in Figure 3. Each test protein can detect the endocytosis signal in TF-1 cells. ADC-2 and hu25G7 have comparable endocytosis activity, with an endocytosis rate of about 70% after 3 hours, while 25G7-Fab has a faster endocytosis and a higher endocytosis rate. Compared with AZD1402-TAG, ADC-2, 25G7-Fab and hu25G7 have significantly better endocytosis speed and endocytosis rate than AZD1402-TAG, and become stable after about 2 hours. ADC-2 has a slightly weaker endocytosis activity than 25G7-Fab.

[0281] Test Example 5: Pharmacokinetic study of antibody-drug conjugate ADC-2 in a mouse model 1. Test protein Antibody-drug conjugate ADC-2

[0282] 2. Experimental method 2.1 Experimental animals C57BL6 / J mouse, 8 weeks old, male, Saiye (Suzhou) Biological Technology Co., Ltd. 2.2 Administration to mice Mice were administered intratracheal aerosol (intratracheal, it), and 300-400μL of blood was collected from the mandible at different times after administration. After standing for 2 hours, the blood was centrifuged at 7500 rpm, 4℃ for 10 min to collect serum. After blood collection, the mice were anesthetized and fixed, the neck muscles of the mice were bluntly peeled to expose the trachea, the trachea was opened laterally, the lavage needle was slowly inserted, and the previously collected 4℃ pre-cooled saline was slowly pushed into the lung tissue to wash it, and the alveolar lavage fluid (BALF) was collected. A total of three washes were performed, the first two with 300μL saline, and the third with 400μL. The collected alveolar lavage fluid was centrifuged at 1000 rpm, 4℃ for 10 min, and the supernatant was collected. After the mice were euthanized, the mouse thoracic cavity was dissected, and the intact mouse lung tissue was isolated and collected. The blood stains on the surface of the lung tissue were washed off with PBS, and the PBS on the surface was wiped off with a paper towel, and the lung tissue was weighed. The lung tissue was placed in a 2 mL centrifuge tube, 1 mL of cell lysis solution (CST, #9803) was added, and the tissue was polished for 2 min at 60 Hz with a homogenizer (Shanghai Jing Xin, Tissuelyser-48), and then centrifuged at 10,000 × g and 4 ° C for 10 min to obtain the supernatant as the lung tissue homogenate, and the protein concentration of the homogenate was detected (Pierce, 23227). The serum, BALF, and lung tissue homogenate were stored at -80 ° C. The administration schedule is shown in Table 4, and the sampling schedule is shown in Table 5.

[0283] [Table 6]

[0284] [Table 7]

[0285] 2.3 Detection of ADC-2 content (1) Coating: The antigen hIL-4Rα was diluted to 1 μg / mL with carbonate buffer (Acro, ILR-H5221), and 100 μL was added to each well of the ELISA plate and incubated overnight at 4° C. (16 to 18 h). (2) Plate washing: The ELISA plate was allowed to recover to room temperature, and 300 μL of 0.05% PBST (PBS+0.05% Tween 20) buffer was added to each well to wash the plate, washing the plate a total of three times. (3) Blocking: 300 μL of PBS containing 3% BSA was added to each well and incubated at room temperature on a shaker at 400 rpm for 2 hours. (4) Washing the plate: 300 μL of 0.05% PBST was added to each well, and the plate was washed three times. (5) Sample incubation: 100 μL of diluted serum, BALF or lung tissue homogenate was added to the ELISA plate and incubated for 1.5 h on a shaker at 400 rpm at room temperature. (6) Washing the plate: 300 μL of 0.05% PBST was added to each well, and the plate was washed three times. (7) Detection: The detection antibody (Invitrogen, A18811) was diluted 10,000-fold with assay buffer (0.05% PBST containing 0.5% BSA) and then added to the ELISA plate at 100 μL / well and incubated at 400 rpm on a shaker at room temperature for 1 h. (8) Washing the plate: 300 μL of 0.05% PBST was added to each well, and the plate was washed three times. (9) Color development: 100 μL of TMB substrate (Sera care, 5120-0080) was added to each well and incubated at room temperature for 15 minutes. (10) Stop: 100 μL of substrate reaction stop solution (Solarbio, C1058) was added to each well, and the light absorption value at a wavelength of 620 nm was detected.

[0286] 2.4 Detection of free budesonide content 30 μL of serum, BALF or lung tissue homogenate was taken, 30.0 μL of internal standard working solution (5.00 ng / mL budesonide-D8) and 120 μL of acetonitrile were added, vortexed for 1 min, centrifuged at 13,000 rpm and 4°C for 5 min, 100 μL of the supernatant was taken and placed in a 96-well plate, 100 μL of 0.3% FA dilution solution was added, incubated at 1,000 rpm on a shaker at room temperature for 15 min, and 20 μL was taken for LC-MS / MS analysis (Shimadzu Japan, Shimadzu Liquid Chromatography System, AB Sciex, Triple Quad 6500). + The analysis was performed using a triple quadrupole tandem mass spectrometer.

[0287] 3. Experimental results The pharmacokinetic parameters of the antibody-drug conjugate ADC-2 and free budesonide are shown in Table 6, and the blood concentration-time curves are shown in Figure 4. [Table 8]

[0288] The experimental results showed that after intratracheal administration of the antibody-drug conjugate ADC-2, the drug was distributed mainly in serum and BALF, with low exposure in serum, and very low exposure in each tissue.

[0289] Test Example 6: In vivo efficacy of ADC-2 in an OVA-induced mouse asthma model 1. Test drug These are antibody-drug conjugates ADC-2, 25G7-Fab, AZD1402-TAG, and Budesonide.

[0290] 2. Experimental method 2.1 Experimental animals IL-4 hu / hu-IL-4Rα hu / hu double transgenic C57BL6 / J mouse, 8 weeks old, female, Saiye (Suzhou) Biological Technology Co., Ltd. 2.2 Administration to mice The animals were randomly divided into normal control animals and model-constructed animals. On days 0, 7, and 14, 100 μL of PBS solution containing 200 μg of OVA (Sigma, A5503) was intraperitoneally injected, respectively, and the mice were sensitized using an equal volume of aluminum hydroxide as an adjuvant. On days 21 to 27, the mice were inhaled with a 3% OVA nebulized solution once a day for 35 min / time. From day 21, the mice were administered intratracheal nebulization 0.5 hours before inhaling the OVA nebulized solution. The specific administration schedule is shown in Table 7. On day 28, the mice were euthanized, and the neck muscles of the mice were bluntly dissected to expose the trachea, which was opened laterally, and the lavage needle was slowly inserted, and the previously collected 4°C pre-cooled saline was slowly pushed into the lung tissue to wash and collect the alveolar lavage fluid. A total of three lavages were performed, the first two with 300 μL saline, and the third with 400 μL. After centrifugation at 1,000 rpm and 4°C for 10 min, the cell pellet was resuspended in 350 μL pre-cooled saline, and the white blood cells and differential cells were counted using a fully automated hematology analyzer.

[0291] [Table 9]

[0292] 2.3 Experimental Indicators The number of leukocytes and differential cells in BALF. 2.4 Statistical analysis Unless otherwise specified, comparisons between cell numbers of two groups were performed using one-way ANOVA test, and a value of p<0.05 was defined as a statistically significant difference.

[0293] 3. Experimental results The experimental results are shown in Figure 5. Compared with the model construction group, at equimolar doses, the antibody-drug conjugate ADC-2 significantly reduced the number of leukocytes, eosinophils and neutrophils in BALF, and slightly suppressed the number of monocytes, showing superior efficacy to AZD1402-TAG and obviously superior efficacy to 25G7-Fab and Budesonide. ADC-2 shows synergistic efficacy with 25G7-Fab and Budesonide.

[0294] Test Example 7: Dose-finding of in vivo efficacy of ADC-2 in OVA-induced mouse asthma model 1. Test drug These are antibody-drug conjugates ADC-2, AZD1402-TAG, and Budesonide.

[0295] 2. Experimental method 2.1 Experimental animals IL-4 hu / hu-IL-4Rα hu / hu double transgenic C57BL6 / J mouse, 8 weeks old, female, Saiye (Suzhou) Biological Technology Co., Ltd. 2.2 Administration to mice The administration and BALF sampling procedures were the same as in Test Example 6. The specific administration schedule is shown in Table 8.

[0296] [Table 10]

[0297] 2.3 Experimental Indicators The number of leukocytes and differential cells in BALF. 2.4 Statistical analysis Unless otherwise specified, comparisons between cell numbers of two groups were performed using one-way ANOVA test, and a value of p<0.05 was defined as a statistically significant difference.

[0298] 3. Experimental results The experimental results are shown in Figure 6. Compared with the model construction group, 10 μg of ADC-2 can significantly reduce the number of eosinophils in alveolar lavage fluid, which is equivalent to AZD1402-TAG and slightly better than budesonide, which is much higher than the clinical equivalent dose. In addition, 10 μg of ADC-2 also has a very significant inhibitory ability on the number of white blood cells, and also inhibits the number of monocytes and neutral granulocytes to a certain extent. These results show that 1 / 10 molar dose of ADC-2 can achieve the efficacy equivalent to AZD1402-TAG.

Claims

1. Formula (I): 【Chemistry 1】 wherein Ab is an anti-IL-4R antibody or an antigen-binding fragment thereof; L is a linker covalently linking Ab to D, and k is 1 to 20; D is a glucocorticoid or a residue thereof, and the glucocorticoid is selected from budesonide or ciclesonide. The antibody-drug conjugate shown in

2. D is 【Chemistry 2】 The antibody-drug conjugate of claim 1, wherein

3. The anti-IL-4R antibody or antigen-binding fragment thereof is selected from the following (I) to (IV): (I) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 set forth in SEQ ID NO:38, SEQ ID NO:7 and SEQ ID NO:40, respectively; (II) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 set forth in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively; (III) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, respectively; (IV) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 set forth in SEQ ID NO:42, SEQ ID NO:39 and SEQ ID NO:8, respectively; The antibody-drug conjugate of claim 1, comprising any one selected from the following:

4. the anti-IL-4R antibody is any one selected from a mouse antibody, a chimeric antibody, a fully human antibody, and a humanized antibody; Preferably, the anti-IL-4R antibody or antigen-binding fragment thereof comprises a FR derived from the human germline light chain IGKV3-11*01 or a FR having at least 95% identity thereto, wherein the FR preferably comprises a backmutation, the backmutation being one or more selected from 46P, 47W, and 71Y; and / or the FR comprises a FR derived from human germline heavy chain IGHV3-48*01 or a FR having at least 95% identity thereto, wherein the FR preferably comprises a backmutation, the backmutation being one or more selected from 49A, 67S, and 93T; Alternatively, preferably, the anti-IL-4R antibody or antigen-binding fragment thereof comprises a FR derived from the human germline light chain IGKV2D-29*01 or a FR having at least 95% identity thereto, wherein the FR preferably comprises a backmutation, the backmutation being selected from 4L and / or 58I, and / or The present invention comprises a FR derived from human germline heavy chain IGHV1-2*02 or a FR having at least 95% identity thereto, wherein the FR preferably contains a back mutation, and the back mutation is one or more selected from 69L, 71I, 73K, and 94K. The antibody-drug conjugate of claim 1.

5. The anti-IL-4R antibody or antigen-binding fragment thereof has the following (i) to (vi): (i) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 43 or an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to SEQ ID NO: 43; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 37 or an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to SEQ ID NO: 37; (ii) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 9 or an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to SEQ ID NO: 9; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to SEQ ID NO: 10; (iii) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to SEQ ID NO: 1; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to SEQ ID NO: 2; (iv) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 43 or an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to SEQ ID NO: 43; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 41 or an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to SEQ ID NO: 41; (v) a heavy chain variable region comprising a sequence set forth in one of SEQ ID NOs: 25-27 or a sequence having at least 90%, 95%, 98%, or 99% identity to one of SEQ ID NOs: 25-27; and a light chain variable region comprising a sequence set forth in one of SEQ ID NOs: 28-30 or a sequence having at least 90%, 95%, 98% or 99% identity to one of SEQ ID NOs: 28-30; (vi) a heavy chain variable region comprising a sequence set forth in one of SEQ ID NOs: 31-33 or a sequence having at least 90%, 95%, 98%, or 99% identity to one of SEQ ID NOs: 31-33; and a light chain variable region comprising a sequence set forth in one of SEQ ID NOs: 34-36 or a sequence having at least 90%, 95%, 98% or 99% identity to one of SEQ ID NOs: 34-36; and Preferably, the anti-IL-4R antibody or antigen-binding fragment thereof is selected from the group consisting of (vii) to (xii) below, namely: (vii) a heavy chain variable region whose sequence is set forth in SEQ ID NO: 43, and a light chain variable region whose sequence is set forth in SEQ ID NO: 37; (viii) a heavy chain variable region whose sequence is set forth in SEQ ID NO: 9, and a light chain variable region whose sequence is set forth in SEQ ID NO: 10; (ix) a heavy chain variable region whose sequence is represented by SEQ ID NO: 1, and a light chain variable region whose sequence is represented by SEQ ID NO: 2; (x) a heavy chain variable region whose sequence is represented by SEQ ID NO: 43, and a light chain variable region whose sequence is represented by SEQ ID NO: 41; (xi) a heavy chain variable region whose sequence is set forth in one of SEQ ID NOs: 25 to 27, and a light chain variable region whose sequence is set forth in one of SEQ ID NOs: 28 to 30; (xii) a heavy chain variable region whose sequence is set forth in one of SEQ ID NOs: 31 to 33, and a light chain variable region whose sequence is set forth in one of SEQ ID NOs: 34 to 36; The antibody-drug conjugate of claim 1, comprising any one selected from the following:

6. the anti-IL-4R antibody or antigen-binding fragment thereof comprises a heavy chain constant region, wherein the heavy chain constant region is a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region or a variant thereof; and / or the antigen-binding fragment is Fab, Fv, scFv, Fab', or F(ab')2; The antibody-drug conjugate of claim 1.

7. The anti-IL-4R antibody or antigen-binding fragment thereof comprises (a) to (d), i.e., (a) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 44, or an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to SEQ ID NO: 44; and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 45 or an amino acid sequence having at least 90%, 95%, 98% or 99% identity to SEQ ID NO: 45; (b) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 19 or an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to SEQ ID NO: 19; and a light chain comprising the amino acid sequence set forth in SEQ ID NO:20 or an amino acid sequence having at least 90%, 95%, 98% or 99% identity to SEQ ID NO:20; (c) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 17 or an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to SEQ ID NO: 17; and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 18 or an amino acid sequence having at least 90%, 95%, 98% or 99% identity to SEQ ID NO: 18; (d) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 44, or an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to SEQ ID NO: 44; and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 46 or an amino acid sequence having at least 90%, 95%, 98% or 99% identity to SEQ ID NO: 46; and More preferably, the anti-IL-4R antibody or antigen-binding fragment thereof comprises (e) to (i), i.e., (e) a heavy chain having the sequence set forth in SEQ ID NO: 44 and a light chain having the sequence set forth in SEQ ID NO: 45; (f) a heavy chain having the sequence set forth in SEQ ID NO: 47 and a light chain having the sequence set forth in SEQ ID NO: 45; (g) a heavy chain having the sequence set forth in SEQ ID NO: 17 and a light chain having the sequence set forth in SEQ ID NO: 18; (h) a heavy chain having the sequence set forth in SEQ ID NO: 19 and a light chain having the sequence set forth in SEQ ID NO: 20; (i) a heavy chain having the sequence set forth in SEQ ID NO: 44 and a light chain having the sequence set forth in SEQ ID NO: 46; The antibody-drug conjugate of claim 1, comprising any one selected from the following:

8. The anti-IL-4R antibody or antigen-binding fragment thereof comprises (a) to (f), i.e., (a) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 48, or an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to SEQ ID NO: 48; and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 18 or an amino acid sequence having at least 90%, 95%, 98% or 99% identity to SEQ ID NO: 18; (b) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 49 or an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to SEQ ID NO: 49; and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 18 or an amino acid sequence having at least 90%, 95%, 98% or 99% identity to SEQ ID NO: 18; (c) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 50 or an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to SEQ ID NO: 50; and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 45 or an amino acid sequence having at least 90%, 95%, 98% or 99% identity to SEQ ID NO: 45; (d) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 50, or an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to SEQ ID NO: 50; and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 46 or an amino acid sequence having at least 90%, 95%, 98% or 99% identity to SEQ ID NO: 46; (e) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 51, or an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to SEQ ID NO: 51; and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 45 or an amino acid sequence having at least 90%, 95%, 98% or 99% identity to SEQ ID NO: 45; (f) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 51, or an amino acid sequence having at least 90%, 95%, 98%, or 99% identity to SEQ ID NO: 51; and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 46 or an amino acid sequence having at least 90%, 95%, 98% or 99% identity to SEQ ID NO: 46; and More preferably, the anti-IL-4R antibody or antigen-binding fragment thereof is selected from (h) to (m), i.e., (h) a heavy chain having the sequence set forth in SEQ ID NO: 48 and a light chain having the sequence set forth in SEQ ID NO: 18; (i) a heavy chain having the sequence set forth in SEQ ID NO: 49 and a light chain having the sequence set forth in SEQ ID NO: 18; (j) a heavy chain having the sequence set forth in SEQ ID NO: 50 and a light chain having the sequence set forth in SEQ ID NO: 45; (k) a heavy chain having the sequence set forth in SEQ ID NO: 50 and a light chain having the sequence set forth in SEQ ID NO: 46; (l) a heavy chain having the sequence set forth in SEQ ID NO: 51 and a light chain having the sequence set forth in SEQ ID NO: 45; (m) a heavy chain having the sequence set forth in SEQ ID NO: 51, and a light chain having the sequence set forth in SEQ ID NO: 46; The antibody-drug conjugate of claim 1, comprising any one selected from the following:

9. L-D is represented by the following formula: 【Transformation 3】 wherein Str is a stretch unit covalently attached to Ab; Sp is a spacer unit, Pep can be an amino acid unit, a disulfide moiety, a sulfonamide moiety or the following non-peptide chemical moieties: 【Chemistry 4】 wherein W is —NH-heterocycloalkylene- or heterocycloalkyl group, and Y is a heteroarylene group, an arylene group, —C(O)C １－６ Alkylene group, C ２－６ Alkenylene group, C １－６ Alkylene group or —C １－６ alkylene-NH-, and each R １６ is independently C １－６ Alkyl group, C ２－６ alkenyl group, -(C １－６ alkylene)NHC(NH)NH ２ or -(C １－６ (alkylene)NHC(O)NH ２ Selected from R １７ and R １８ are each independently hydrogen, C １－６ Alkyl group, C ２－６ is selected from an alkenyl group, an aryl group, and a heteroaryl group, or R １７ and R １８ Let's go together ３－６ A cycloalkyl group can be formed, and R １９ and R ２０ are each independently C １－６ Alkyl group, C ２－６ Alkenyl groups, aryl groups, heteroaryl groups, (C １－６ alkyl)OCH ２ - or R １９ and R ２０ Let's go together ３－６ can form a cycloalkyl ring] 2. The antibody-drug conjugate of claim 1, wherein the chemical moiety is

10. Str is 【Transformation 5】 Among these, R ２１ Ha, -W １ -C(O)-, -C(O)-W １ -C(O)-, -(CH ２ CH ２ O) ｐ１ C(O)-,-(CH ２ CH ２ O) ｐ１ CH ２ C(O)-,-(CH ２ CH ２ O) ｐ１ CH ２ CH ２ C(O)-, among which W １ is C １－６ Alkylene group, C １－６ alkylene-cycloalkyl groups or straight-chain heteroalkyl groups of 1 to 8 atoms, wherein the heteroalkyl groups contain 1 to 3 heteroatoms selected from N, O, or S, wherein the alkyl groups, cycloalkyl groups, and straight-chain heteroalkyl groups each independently optionally contain halogen, deuterium, hydroxyl, cyano, amino, C １－６ Alkyl group, halo C １－６ Alkyl group, deuterated C １－６ Alkyl group, C １－６ Alkoxy group and C ３－６ cycloalkyl groups, and each p1 independently represents an integer of 1 to 20; L １ is -NR ２２ - (CH ２ CH ２ O) ｐ２ CH ２ CH ２ C(O)-, -NR ２２ - (CH ２ CH ２ O) ｐ２ CH ２ C(O)-, -S-(CH ２ ) ｐ２ C(O)-,-(CH ２ ) ｐ２ C(O)— or a single bond, preferably a single bond; p2 is independently an integer of 1 to 20; R ２２ is a hydrogen atom, C １－６ Alkyl group, halo C １－６ Alkyl group or deuterated C １－６ selected from alkyl groups, The antibody-drug conjugate of claim 9.

11. R ２１ is -C １－６ Alkylene C(O)-, -(CH ２ -CH ２ O) ２ C(O)-,-(CH ２ -CH ２ O) ２ CH ２ C(O)-,-(CH ２ -CH ２ O) ２ CH ２ CH ２ C(O)-,-(CH ２ -CH ２ O) ３ C(O)- and -(CH ２ -CH ２ O) ４ The antibody-drug conjugate of claim 10, wherein the C(O)- is selected from the group consisting of C(O)-, ...

12. The antibody-drug conjugate of claim 10, wherein the Pep is selected from valine-citrulline, alanine-alanine-asparagine, glycine-glycine-lysine, valine-lysine (Val-lys), valine-alanine, valine-phenylalanine, and glycine-glycine-phenylalanine-glycine.

13. Spは、-NHCH ２ -、-NHCH ２ CH ２ -、-NHCH ２ CH ２ CH ２ -、-NHCH ２ -O-CH ２ -、-NHCH ２ CH ２ -O-CH ２ -、-NH(CH ２ ) ３ -C(O)-、-NHCH ２ -O-CH ２ -C(O)-, -NH(CH) ２ ) ２ -O-CH ２ -C(O)-, 【Transformation 6】 The antibody-drug conjugate of claim 10, selected from the group consisting of

14. The linker L in the antibody-drug conjugate is maleimide-(PEG). ４ -CH ２ CH ２ C(O)-Gly-Gly-Phe-Gly, maleimide-(PEG) ２ -Val-Cit, Maleimide-(PEG) ６ -Val-Cit, Maleimide-(PEG) ８ -Val-Cit, Maleimide-(PEG) ４ -CH ２ CH ２ C(O)-Val-lys, maleimide-(CH ２ ) ５ -Val-Cit, maleimide-(CH ２ ) ５ -Val-lys, maleimide-(CH ２ ) ５ -Gly-Gly-Phe-Gly, maleimide-(PEG) ４ -CH ２ CH ２ C(O)-Gly-Gly-Phe-Gly, maleimide-(PEG) ２ -Ala-Ala-Asn, maleimide-(PEG) ６ -Ala-Ala-Asn, maleimide-(PEG) ８ -Ala-Ala-Asn, maleimide-(PEG) ４ -triazole-(PEG) ３ -sulfonamide, maleimide-(PEG) ２ -CH ２ CH ２ C(O)-Val-lys, maleimide-(PEG) ４ -triazole-(PEG) ３ -sulfonamide or Mal-(PEG) ４ -triazole-(PEG) ３ The antibody-drug conjugate of claim 1, comprising a -disulfide.

15. The following formula: 【Transformation 7】 wherein k is selected from 1 to 10, p1 is selected from 2, 4, 6, or 8, and p3 and p4 are each independently selected from 0, 1, or 2; or 【Transformation 8】 [In the formula, k is selected from 1 to 10, p1 is selected from 2, 4, 6, or 8, and p3 and p4 are each independently selected from 0, 1, or 2] The antibody-drug conjugate of claim 1, wherein 【Request Item 16】 【Chemistry 9】 where k is selected from 1 to 10; The antibody-drug conjugate of claim 15.

17. Formula II-A: 【Chemistry 10】 wherein p1 is selected from 2, 4, 6, or 8; p3 and p4 are each independently selected from 0, 1, or 2; and D is budesonide or ciclesonide. or a medicinal salt thereof.

18. Formula II-B: 【Chemistry 11】 wherein X is a halogen, preferably bromine; p1 is selected from 2, 4, 6, or 8; p3 and p4 are each independently selected from 0, 1, or 2; and D is budesonide or ciclesonide. or a medicinal salt thereof.

19. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 1 to 16 and a pharmaceutically acceptable excipient.

20. Formula (I): 【Chemistry 12】 [wherein Ab is an anti-IL-4R antibody or an antigen-binding fragment thereof, preferably an anti-IL-4R Fab fragment; L is a linker covalently linking Ab to D, and k is 1 to 20; D is a glucocorticoid or a residue thereof. An inhalable pharmaceutical composition comprising an antibody-drug conjugate represented by the formula (I) and a pharmaceutically acceptable vector.

21. 20. The pharmaceutical composition according to claim 19, for treating or preventing an immune disease or condition, wherein the disease or condition is preferably asthma, nasal polyps, chronic sinusitis, allergic skin disease, eosinophilic esophagitis, chronic obstructive pulmonary disease, allergic rhinitis, arthritis, inflammatory disease, allergic reaction, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis disease, or kidney disease, more preferably asthma or allergic skin disease.

22. A pharmaceutical composition according to claim 20 for treating or preventing an immune disease or condition, wherein the disease or condition is preferably asthma, nasal polyps, chronic sinusitis, allergic skin disease, eosinophilic esophagitis, chronic obstructive pulmonary disease, allergic rhinitis, arthritis, inflammatory disease, allergic reaction, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis and kidney disease, and more preferably asthma or allergic skin disease.

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