WO2023143351A1 - 糖皮质激素的药物偶联物 - Google Patents

糖皮质激素的药物偶联物 Download PDF

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WO2023143351A1
WO2023143351A1 PCT/CN2023/073057 CN2023073057W WO2023143351A1 WO 2023143351 A1 WO2023143351 A1 WO 2023143351A1 CN 2023073057 W CN2023073057 W CN 2023073057W WO 2023143351 A1 WO2023143351 A1 WO 2023143351A1
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seq
antibody
amino acid
acid sequence
heavy chain
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PCT/CN2023/073057
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English (en)
French (fr)
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任文明
祝令建
陈豪
唐满平
林�源
周彩红
黄建
廖成
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上海盛迪医药有限公司
上海森辉医药有限公司
江苏恒瑞医药股份有限公司
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Publication of WO2023143351A1 publication Critical patent/WO2023143351A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators

Definitions

  • the disclosure belongs to the field of medicine, in particular to a drug conjugate of glucocorticoid.
  • Interleukin-4 consists of 153 amino acids with a molecular weight of about 17 kDa. Initially, IL-4 was discovered because of its ability to stimulate B cell proliferation and was named B cell stimulating 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 4 ⁇ helices. IL-4 is secreted by TH2 cells, participates in TH2-mediated immune response, and has a wide range of biological activities, including stimulating the proliferation of T cells, mast cells, granulocytes, megakaryocytes, and erythrocytes.
  • BSF-1 B cell stimulating factor-1
  • IL-4 can also stimulate B cells to express major histocompatibility complex class 2 molecules.
  • IL-13 and IL-4 have approximately 30% amino acid sequence homology and many similar functions. Both IL-4 and IL-13 can promote B cell proliferation and co-stimulate CD40/CD40L to induce IgM type conversion to IgE.
  • IL-4 promotes the aggregation of mast cells, up-regulates the expression of high-affinity IgE receptors on mast cells and IgE low-affinity receptor CD23 (Fc ⁇ RII) on B cells, up-regulates the expression of vascular endothelial cell adhesion molecule (VCAM-1), and promotes the expression of eosinophils. , T lymphocytes, monocytes and basophils transfer. Unlike IL-13, IL-4 can promote the differentiation of naive T cells into TH2.
  • IL-4 needs to bind to membrane receptors to exert biological functions.
  • the human interleukin receptor (IL-4R) is a heterodimer formed by two polypeptide chains, one of which has a high affinity for IL-4, because the IL-4R ⁇ chain has a strong affinity for IL-4 in the IL-4R complex.
  • the combination of -4 plays a leading role, so IL-4R ⁇ is often used instead of IL-4R in many scientific studies and reports.
  • IL-4R is expressed on various cells such as human B cells, mast cells, eosinophils, basophils, macrophages/monocytes, DC cells, fibroblasts, airway epithelium and smooth muscle.
  • IL-4R ⁇ can form two types of receptor complexes with other subunits, and the type I receptor composed of IL-4R ⁇ and ⁇ c is mainly expressed in hematopoietic stem cells.
  • IL-4 mainly acts through the type II receptor composed of IL-4R ⁇ and IL-13R ⁇ 1.
  • Type II receptors are co-receptors for IL-4 and IL-13, and IL-13 functions in combination with IL-13R ⁇ 1. Both type I receptors and type II receptors transduce signals through the Jak/STAT pathway.
  • IL-4R ⁇ , ⁇ c and IL-13R ⁇ 1 bind to Jak1, Jak3 and Tyk2 respectively to activate downstream pathways.
  • IL-4 and IL-13 can also pass through The insulin receptor substrate family (IRS) transduces signals and finally activates PI3-K and NF- ⁇ B in the nucleus.
  • IFS insulin receptor substrate family
  • Blocking IL-4R can inhibit both IL-4 and IL-13 biological functions.
  • Atopic dermatitis also known as atopic dermatitis or atopic dermatitis
  • AD Atopic dermatitis
  • IL-4, IL-5, IL-10 and IL-13 are a common disease in dermatology. It is more common in children and adolescents.
  • TH2 factors are related to the disease process of AD, and the overexpression of IL-4, IL- Mice with 13 and other TH2 factors exhibit skin protection defects and AD-like symptoms [14][15] .
  • IL-13 and IL-4 also play an important role in asthma.
  • Asthma is a common lung inflammatory disease characterized by airway hyperresponsiveness (AHR), mucus hypersecretion, fibrosis, and elevated IgE levels.
  • Nonspecific stimuli such as cold air often Leading to increased airway hyperresponsiveness, AHR and mucus hypersecretion lead to airway obstruction, which is the main cause of death in asthma.
  • TH2 factor plays an important role in the process of asthma, and IL-4 and IL-13 are overexpressed in bronchial and alveolar lavage fluid of asthmatic patients.
  • IL-13 and IL-4 share some functional similarities, some studies have shown that IL-13 plays a more important role than other Th2 cytokines in the progression of asthma.
  • IL-13 can promote the differentiation and fibrosis of goblet cells. Injection of recombinant IL-13 into the airways of allergen-na ⁇ ve mice resulted in airway inflammation, mucus hypersecretion, and airway hyperresponsiveness, and injection of soluble IL13R ⁇ 2 prevented AHR, mucus hypersecretion, and lung inflammation in mice happened. Injection of IL-4R ⁇ antibody in an asthma model can reduce eosinophils in AHR and alveolar lavage fluid. Research suggests that blocking IL-4R ⁇ may be effective in treating asthma.
  • Glucocorticoids are also more effective drugs for the treatment of allergic diseases and inflammation.
  • glucocorticoids produced in vivo such as cortisol and corticosterone
  • synthetic glucocorticoids such as dexamethasone, prednisone, prednisolone, and budesonide are known. Since these glucocorticoids have a steroid structure, they are collectively referred to as steroids and are used in the treatment of various diseases. However, these steroids may cause side effects such as steroid peptic ulcer, steroid purpura, steroid pancreatitis, steroid diabetes, steroid cataract, and steroid glaucoma due to their use.
  • ADC Antibody drug conjugate
  • ADC refers to a monoclonal antibody or antibody fragment linked to a biologically active drug through a stable chemical linker compound.
  • Most ADCs in preclinical and clinical development are used in oncology indications, where the cytotoxic payload targets antigen-expressing cancer cells.
  • modulation of pathogenic cellular activity via ADC-mediated delivery of bioactive small molecules is also attractive for non-oncology indications, leading to widespread adoption of this technology.
  • glucocorticoids Some drug conjugates of glucocorticoids have been disclosed in the prior art, such as WO2017210471, WO2019106609, WO2019136487, etc.
  • ADC antibody-drug conjugate
  • Ab is an anti-IL-4R antibody or an antigen-binding fragment thereof
  • L is a linker covalently linking Ab to D
  • 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 value between any two values)
  • D is a glucocorticoid or a residue thereof, and the glucocorticoid is selected from dexamethasone, prednisone, prednisolone, budesonide, mometasone, beclomethasone dipropionate, fluticasone, triamcinolone acetonide and cyclic
  • a sononide for example may be budesonide or ciclesonide.
  • D is represented by the following formula:
  • anti-IL-4R antibodies or antigen-binding fragments thereof may be known, such as anti-IL described in, for example, WO2010053751 , WO2001092340 , WO2008054606 , WO2014031610 , WO2020038454 (each of which is incorporated herein by reference) -4R antibody or antigen-binding fragment thereof.
  • 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.
  • the anti-IL-4R antibody or antigen-binding fragment thereof is an anti-human IL-4R antibody or antigen-binding fragment thereof.
  • 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:
  • And/or antibody light chain variable regions comprising:
  • Table 1 shows the CDR sequences of anti-IL-4R antibodies or antigen-binding fragments thereof.
  • the anti-IL-4R antibody or antigen-binding fragment thereof comprises any one of the following (I) to (IV):
  • 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;
  • 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;
  • Light chain variable region which comprises respectively shown in SEQ ID NO: 38, SEQ ID NO: 7 and SEQ ID NO: 40 LCDR1, LCDR2 and LCDR3;
  • 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.
  • 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.
  • the anti-IL-4R antibody or antigen-binding fragment thereof comprises a FR sequence derived from human germline light chain IGKV3-11*01 (SEQ ID NO: 22, used for antibody 25G7) or A sequence with a back mutation that is at least 95% identical to it.
  • the back mutation is selected from one or more of L46P, L47W, and F71Y.
  • 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, used for antibody 25G7) Or a sequence with back mutations at least 95% identical thereto.
  • the back mutation is selected from one or more of S94A, F67S, and A93T.
  • 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, used for antibody 7B10) Or a backmutated sequence having at least 95% identity therewith.
  • the back mutation is selected from M4L and/or V58I.
  • 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, used for antibody 7B10) Or a backmutated sequence having at least 95% identity therewith.
  • the back mutation is selected from one or more of M69L, R71I, T73K, and R94K.
  • 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 contains:
  • the heavy chain variable region of the anti-IL-4R antibody or antigen-binding fragment is shown in the sequence SEQ ID NO: 1, and the light chain variable region is shown in the sequence SEQ ID NO: 2; or
  • variable region of the heavy chain is shown in the sequence SEQ ID NO: 9
  • variable region of the light chain is shown in the sequence SEQ ID NO: 10; or
  • variable region of the heavy chain is shown in the sequence of SEQ ID NO: 43, and the variable region of the light chain is shown in the sequence of 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.
  • 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 contains:
  • the heavy chain variable region is as shown in one of the sequences of SEQ ID NO:25-27, and the light chain variable region is as shown in one of the sequences of SEQ ID NO:28-30;
  • the heavy chain variable region is shown in one of the sequences SEQ ID NO: 31-33, and the light chain variable region is shown in one of the sequences SEQ ID NO: 34-36.
  • 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 variants thereof. In some specific schemes, the heavy chain constant region of human IgG1 or its variants are included. In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof comprises the constant region of human ⁇ , ⁇ chain or variants thereof.
  • the anti-IL-4R antibody or antigen-binding fragment thereof wherein the antibody is a humanized antibody
  • the heavy chain sequence is as shown in SEQ ID NO: 17 or has at least 85% sequence identity therewith
  • the light chain sequence is as set forth in SEQ ID NO: 18 or has at least 85% sequence identity thereto.
  • the anti-IL-4R antibody or antigen-binding fragment thereof wherein the antibody is a humanized antibody
  • the heavy chain sequence is as shown in SEQ ID NO: 19 or has at least 85% sequence identity therewith
  • the light chain sequence is as set forth in SEQ ID NO: 20 or has at least 85% sequence identity thereto.
  • the anti-IL-4R antibody or antigen-binding fragment thereof wherein the antibody is a humanized antibody
  • the heavy chain sequence is as shown in SEQ ID NO: 44 or has at least 85% sequence identity therewith
  • the light chain sequence is as set forth in SEQ ID NO: 45 or has at least 85% sequence identity thereto.
  • the anti-IL-4R antibody or antigen-binding fragment thereof wherein the antibody is a humanized antibody
  • the heavy chain sequence is as shown in SEQ ID NO: 44 or has at least 85% sequence identity therewith
  • the light chain sequence is as set forth in SEQ ID NO: 46 or has at least 85% sequence identity thereto.
  • an isolated anti-IL-4R antibody or antigen-binding fragment thereof characterized in that it competes with any anti-IL-4R antibody or antigen-binding fragment thereof as described above for binding to human IL-4R or its epitope.
  • a bispecific antibody or a multispecific antibody comprising any of the above-mentioned anti-IL-4R antibodies or antigen-binding fragments thereof light chain variable region and/or heavy chain variable region .
  • a single chain antibody comprising the light chain variable region and/or the heavy chain variable region of any anti-IL-4R antibody or antigen-binding fragment thereof as described above.
  • 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.
  • a human IgG2 or IgG4 heavy chain constant region is included. Because IgG2 or IgG4 has no ADCC toxicity.
  • IgG1 without ADCC antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity
  • the variant comprises a heavy chain constant region mutation with reduced or absent ADCC effector function, such as, but not limited to, N297A, L234A, L235A of IgGl.
  • IgG1 comprises mutations E239D and M241L.
  • anti-IL-4R antibodies or antigen-binding proteins thereof of the present disclosure are coded according to Kabat.
  • the anti-IL-4R antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises:
  • 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 set forth in SEQ ID NO: 19, and the light chain sequence is set forth in SEQ ID NO: 20; or
  • the heavy chain sequence is set forth in SEQ ID NO: 44, and the light chain sequence is set forth in SEQ ID NO: 45; or
  • the heavy chain sequence is set forth in SEQ ID NO: 44, and the light chain sequence is set forth 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.
  • the anti-IL-4R antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises:
  • 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 set forth in SEQ ID NO: 49, and the light chain sequence is set forth in SEQ ID NO: 18; or
  • the heavy chain sequence is set forth in SEQ ID NO: 50, and the light chain sequence is set forth in SEQ ID NO: 45; or
  • the heavy chain sequence is set forth in SEQ ID NO: 50, and the light chain sequence is set forth in SEQ ID NO: 46; or
  • the heavy chain sequence is set forth in SEQ ID NO: 51, and the light chain sequence is set forth 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.
  • the anti-IL-4R antibody or antigen-binding fragment thereof may be an antibody variant having 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid changes, and/or 1 to 10 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid changes in the heavy chain.
  • the above-mentioned variant has the same or similar biological function or effect as the parental anti-IL-4R antibody or fragment thereof.
  • antigen-binding fragments include, but are not limited to, Fab, Fab', Fv, F(ab')2, linear antibody, scFv (single chain Fv antibody), tandem di-scFv, tandem tri-scFv, double chain Antibody (diabody), triabody (triabody), Tetrabody, sdAb (single domain antibody or nanobody), sdFv, peptibody, domain antibody, multispecific antibody (e.g. bispecific, trispecific or tetraspecific) , dsFv (disulfide bond stabilized Fv), ScdsFv (disulfide bond stabilized single chain Fv antibody).
  • the antigen-binding fragment of the anti-IL-4R antibody in the antibody-drug conjugate of the present disclosure or a pharmaceutically acceptable salt or solvate thereof is combined with the antigen of the above-mentioned anti-IL-4R antibody
  • the binding fragments bind the same IL-4R or an epitope thereof.
  • a polynucleotide such as DNA or RNA, encoding an anti-IL-4R antibody or antigen-binding fragment thereof as described above is provided.
  • expression vectors comprising the polynucleotides described above are provided, such as eukaryotic expression vectors, prokaryotic expression vectors, viral vectors.
  • host cells transformed with the above expression vectors such as eukaryotic cells and prokaryotic cells
  • the host cell is bacteria (such as Escherichia coli), yeast (such as Pichia pastoris), mammalian cells (such as Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) 293 cells).
  • bacteria such as Escherichia coli
  • yeast such as Pichia pastoris
  • mammalian cells such as Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) 293 cells.
  • a method for preparing the above-mentioned anti-IL-4R antibody or antigen-binding fragment thereof comprising the steps of: 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 The antibody or antigen-binding fragment thereof.
  • k is any value between 1-10, preferably any value between 2-5. k can be an integer or a decimal.
  • the linker is extracellularly stable such that the ADC remains intact when present in the extracellular environment, but is capable of cleavage when internalized in the cell.
  • the ADC enters a cell expressing an antigen specific for the antibody portion of the ADC
  • the glucocorticoid drug moiety is cleaved from the antibody portion, and the cleavage releases the unmodified form of the glucocorticoid.
  • the cleavable moiety in the linker is a cleavable peptide moiety.
  • ADCs comprising a cleavable peptide moiety exhibit lower levels of aggregation, improved antibody to drug ratio relative to ADCs comprising other cleavable moieties.
  • addition of a cleavable moiety increases cytotoxicity and/or potency relative to a non-cleavable linker.
  • the cleavable peptide moiety is capable of being cleaved by an enzyme, and the linker is one that is cleavable by an enzyme.
  • the enzyme is a cathepsin and the linker is a linker that the cathepsin is capable of cleaving.
  • an enzyme-cleavable linker eg, a cathepsin-cleavable linker
  • a linker comprises a stretcher unit, a chemical moiety fragment covalently linked at one end to the antibody through a carbon atom and at the other end to an amino acid unit, a disulfide moiety, a sulfonamide moiety, or a non-peptidic chemical moiety.
  • Exemplary stretching units include, but are not limited to
  • the linker comprises an amino acid unit, preferably comprising 2 to 7 amino acids selected from the group consisting of phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, Aspartic acid, homolysine, n-methyl-valine, (q is an integer of 1-6) amino acid residues
  • exemplary amino acid units include but not limited to 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 g
  • a linker can comprise at least one polyethylene glycol (PEG) moiety.
  • PEG polyethylene glycol
  • the PEG moiety may for example contain -(PEG) p1- , where p1 is an integer from 1 to 20, for example (PEG) 2 ; (PEG) 4 ; (PEG) 5 .
  • the linker comprises a spacer unit attached to D.
  • the spacer unit comprises p-aminobenzyloxycarbonyl (PAB),
  • the spacer unit comprises p-aminobenzoyl
  • the spacer unit comprises:
  • n1 is an integer selected from 0-6;
  • R 7 is selected from hydrogen, C 1-6 alkyl, -(CH 2 ) n3 -COOH, -(CH 2 ) n4 -OH, n3 is selected from integers between 1-4 , n4 is selected from an integer between 1-6;
  • L b represents -CR 8 (R 9 )-, -O-, -NR 10 - or a single bond, R 8 and R 9 are independently selected from hydrogen, C 1-6 alkane Group, C 3-6 cycloalkyl, -(CH 2 ) n5 -NH 2 , -(CH 2 ) n6 -COOH, -(CH 2 ) n7 -
  • R 8 and R 9 in -NH(CH 2 ) n1 -L a -L b -L c - are independently selected from hydrogen, C 1-6 alkyl, C 3-6 cycloalkyl, for example Hydrogen, methyl, ethyl or cyclopropyl.
  • L a represents -O- or a single bond
  • L b represents -CR 8 (R 9 )- or a single bond
  • R 8 and R 9 form a C 3-6 cycloalkyl group together with the carbon atoms they are connected to.
  • L a represents -O- or a single bond
  • L b represents -CR 8 (R 9 )- or a single bond
  • R 8 and R 9 are independently selected from hydrogen, C 1-6 alkyl, C 3-6 cycloalkyl, -(CH 2 ) n5 -NH 2 , -(CH 2 ) n6 -COOH, -(CH 2 ) n7 -OH
  • R 10 is selected from hydrogen or C 1-6 alkyl
  • n5 is selected from integers between 0-6, n6 is selected from integers between 1-4, n7 is selected from 1 Integer between -4, and when n5 is 0, R 8 and R 9 are different.
  • L a represents -O- or a single bond
  • L b represents -CR 8 (R 9 )- or a single bond
  • L c represents -CH 2 -.
  • L a represents -O- or a single bond
  • L b represents -CR 8 (R 9 )- or a single bond
  • L c represents -CH 2 -
  • R 8 and R 9 are independently selected from hydrogen, C 1-6 alkyl, C 3-6 cycloalkyl, preferably hydrogen, methyl, ethyl or cyclopropyl.
  • -NH(CH 2 ) n1 -L a -L b -L c - in the antibody-drug conjugate is: -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)-,
  • -NH-(CH 2 ) n1 -L a -L b -L c - is -NHCH 2 - or -NHCH 2 CH 2 CH 2 -.
  • the spacer unit in the linker comprises (PEG) 2 .
  • ADCs comprising shorter spacer units (eg , (PEG) 8 ) exhibit lower Aggregation levels and/or higher drug loading.
  • L-D in the antibody conjugate (ADC) of the present disclosure is a chemical moiety represented by the formula:
  • Str is the stretch base unit covalently linked to Ab
  • Pep is selected from amino acid units, disulfide moieties, sulfonamide moieties, or the following non-peptide chemical moieties:
  • W is -NH-heterocycloalkylene- or heterocycloalkyl
  • Y is heteroarylene, arylene, -C(O)C 1-6 alkylene, C 2-6 alkenylene , C 1-6 alkylene or -C 1-6 alkylene-NH-
  • each R 16 is independently selected from C 1-6 alkyl, C 2-6 alkenyl, -(C 1-6 alkylene Base) NHC (NH) NH 2 or - (C 1-6 alkylene) NHC (O) NH 2
  • R 17 and R 18 are each independently selected from hydrogen, C 1-6 alkyl, C 2-6 alkenyl , aryl, heteroaryl, or R 17 and R 18 together can form C 3-6 cycloalkyl
  • R 19 and R 20 are each independently selected from C 1-6 alkyl, C 2-6 alkenyl, aryl , heteroaryl, (C 1-6 alkyl)OCH 2 -, or R 19 and R 20 together can form a
  • Str in the antibody-drug conjugate is selected from chemical moieties represented by the following formula:
  • R 21 is selected from -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)-, wherein W1 is selected from C 1-6 alkylene, C 1-6 alkylene - cycloalkyl or straight chain heteroalkyl of 1 to 8 atoms, said heteroalkyl contains 1 to 3 heteroatoms selected from N, O or S, wherein said alkyl, cycloalkyl and straight chain
  • the heteroalkyl groups are each independently optionally replaced by one or more selected from the group consisting of halogen, deuterium, hydroxyl, cyano, amino, C 1-6 alkyl, halogenated C 1-6 alkyl, deuterated C 1-6 alkyl , C 1-6 alkoxy and C
  • L 1 is selected from -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 an integer from 1 to 20
  • R 22 is selected from a hydrogen atom, C 1-6 alkyl, Halogenated C 1-6 alkyl or deuterated C 1-6 alkyl.
  • R 21 in the antibody-drug conjugate Str is selected from 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 -( CH2 - CH2O ) 4C (O)-.
  • the Pep is selected from the group consisting of valine-citrulline (Val-Cit), alanine-alanine-asparagine (Ala-Ala-Asn), glycine-glycine-lysine Acid (Gly-Gly-lys), Valine-Lysine (Val-lys), Valine-Alanine (Val-Ala), Valine-Phenylalanine (Val-Phe), or Glycine - Glycine-Phenylalanine-Glycine (Gly-Gly-Phe-Gly).
  • the Sp is selected from -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 )-, -NH(CH 2 ) 2 -O-CH 2 -C(O )-,
  • the linker L in the antibody-drug conjugate comprises: 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, Maleyl Imine-(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
  • the antibody-drug conjugate (ADC) of the present disclosure is represented by the following formula:
  • k is selected from 1 to 10 and can be an integer or a decimal
  • p1 is selected from 2, 4, 6 or 8
  • p3 and p4 are each independently selected from 0, 1 or 2;
  • k is selected from 1 to 10 and can be an integer or a decimal
  • p1 is selected from 2, 4, 6 or 8
  • p3 and p4 are each independently selected from 0, 1 or 2;
  • the antibody-drug conjugate (ADC) of the present disclosure is selected from:
  • k is selected from 1 to 10, and can be an integer or a decimal.
  • the present disclosure also provides a compound represented by formula II-A or a pharmaceutically acceptable salt thereof,
  • p1 is selected from 2, 4, 6 or 8, and p3 and p4 are each independently selected from 0, 1 or 2.
  • the present disclosure also provides a compound represented by formula II-B or a pharmaceutically acceptable salt thereof,
  • X is halogen
  • p1 is selected from 2, 4, 6 or 8
  • p3, p4 are each independently selected from 0, 1 or 2.
  • the present disclosure also provides a pharmaceutical composition, comprising at least one aforementioned antibody-drug conjugate, and a pharmaceutically acceptable carrier, diluent or excipient.
  • the unit dose of the pharmaceutical composition is 0.001 mg-1000 mg.
  • the pharmaceutical composition contains 0.01%-99.99% of the aforementioned compound based on the total weight of the composition. In some embodiments, the pharmaceutical composition contains 0.1%-99.9% of the aforementioned compounds. In some embodiments, the pharmaceutical composition contains 0.5%-99.5% of the aforementioned compounds. In some embodiments, the pharmaceutical composition contains 1%-99% of the aforementioned compounds. In some embodiments, the pharmaceutical composition contains 2%-98% of the aforementioned compounds.
  • the pharmaceutical composition contains 0.01%-99.99% of a pharmaceutically acceptable carrier, diluent or excipient based on the total weight of the composition. In some embodiments, the pharmaceutical composition contains 0.1%-99.9% of a pharmaceutically acceptable carrier, diluent or excipient. In some embodiments, the pharmaceutical composition contains 0.5%-99.5% of a pharmaceutically acceptable carrier, diluent or excipient. In some embodiments, the pharmaceutical composition contains 1%-99% of pharmaceutically acceptable carriers, diluents or excipients. In some embodiments, the pharmaceutical composition contains 2%-98% of a pharmaceutically acceptable carrier, diluent or excipient.
  • the antibody-drug conjugates of the disclosure can be administered to a patient in a manner appropriate to the indication, eg, parenterally, topically, or by inhalation.
  • the antagonist can be administered by, for example, intra-articular, intravenous, intramuscular, intralesional, intraperitoneal or subcutaneous routes, by bolus injection or continuous infusion. Local administration at the site of disease or injury is considered transdermal delivery and sustained release from the implant. 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, lozenges, or chewing gum; and topical formulations such as lotions, gels, sprays, and ointments.
  • the present disclosure also provides an inhalable pharmaceutical composition, including the antibody-drug conjugate represented by formula (I) and a pharmaceutically acceptable carrier,
  • Ab is an anti-IL-4R antibody or an antigen-binding fragment thereof
  • L is a linker covalently linking Ab to D
  • 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 value between any two values)
  • D is a glucocorticoid or its residue.
  • the Ab is an anti-IL-4R Fab fragment.
  • the present disclosure also provides the use of the antibody-drug conjugate and/or the pharmaceutical composition comprising the antibody-drug conjugate in the preparation of a medicament for treating or preventing IL-4R-mediated diseases or disorders.
  • the present disclosure also provides the use of the antibody-drug conjugate and/or the pharmaceutical composition comprising the antibody-drug conjugate in the preparation of a medicament for treating or preventing an immune disease or disorder.
  • the disease or condition is selected from the group consisting of: 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. Asthma or allergic skin diseases are preferred.
  • the present disclosure also provides a method of delivering a glucocorticoid to an IL-4R expressing cell, comprising the step of contacting the IL-4R expressing cell with the antibody-drug conjugate of the present disclosure.
  • the present disclosure further provides a kit comprising the antibody-drug conjugate described in the present disclosure, or a pharmaceutical composition.
  • linker refers to a chemical structural fragment or bond that is connected to a ligand at one end and a drug at the other end, and can also be connected after other linkers. Then connect with the drug.
  • a joint may comprise one or more joint components.
  • exemplary linker building blocks include 6-maleimidocaproyl (MC), maleimidopropionyl (MP), valine-citrulline (Val-Cit or vc), alanine-phenyl Alanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), and those derived from coupling with linker reagents: 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).
  • MC 6-maleimidocaproyl
  • MP maleimidopropionyl
  • Vcit valine-citrulline
  • Linkers can include Stretch units, Spacer units, Amino Acid units and Stretcher units. It can be synthesized by methods known in the art, such as described in US2005-0238649A1.
  • the linker can be a "cleavable linker" that facilitates release of the drug in the cell.
  • acid-labile e.g., hydrazone
  • protease-sensitive e.g., peptidase-sensitive
  • photolabile, dimethyl, or disulfide-containing linkers can be used (Chari et al., Cancer Research 52:127-131 (1992); US Patent No. 5,208,020).
  • stretch unit refers to a chemical structural fragment that is covalently linked to the antibody through a carbon atom at one end and to an amino acid unit, disulfide moiety, sulfonamide moiety, or non-peptidic chemical moiety at the other end.
  • spacer unit is a bifunctional compound structural fragment that can be used to couple amino acid units and glucocorticoids to form antibody-drug conjugates. This coupling method can selectively link glucocorticoids to amino acid units superior.
  • amino acid refers to an organic compound containing an amino group and a carboxyl group in the molecular structure, and both the amino group and the carboxyl group are directly connected to the -CH- structure.
  • the general formula is H2NCHRCOOH, R is H, substituted or unsubstituted alkyl, etc. According to the amino linkage
  • the positions of carbon atoms in carboxylic acids can be divided into ⁇ , ⁇ , ⁇ , ⁇ , ⁇ ...-amino acids.
  • amino acids that make up natural proteins have their specific structural characteristics, that is, their amino groups are directly connected to the ⁇ -carbon atom, that is, ⁇ -amino acids, including glycine (Glycine), alanine (Alanine), valine (Valine), Leucine, Isoleucine, Phenylalanine, Tryptophan, Tyrosine, Aspartic acid, Histidine, Asparagine, Glutamic acid, Lysine, Glutamine, Methionine, Arginine , Serine, Threonine, Cysteine, Proline, etc. Unnatural amino acids such as citrulline.
  • antibody-drug conjugate means that a ligand is linked to a biologically active drug through a stable linker unit.
  • antibody drug conjugate refers to linking a monoclonal antibody or antibody fragment with a biologically active glucocorticoid through a stable linker unit. Wherein the antibody or antibody fragment can be combined with the glucocorticoid molecule comprising the linker through a specific group (such as an interchain disulfide bond).
  • drug loading refers to the average amount of drug carried by each antibody-drug conjugate molecule in the antibody-drug conjugate population, and can also be expressed as the ratio of the amount of drug to the amount of antibody.
  • the range of drug loading can be 1-20, preferably 1-10 glucocorticoids (D) linked to each antibody (Ab).
  • the drug loading is expressed as k, which may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or the mean of any two values in between.
  • the average amount of drug per ADC molecule after conjugation can be identified using routine methods such as UV/Vis spectroscopy, mass spectrometry, ELISA assay, monoclonal antibody size variant assay (CE-SDS) and HPLC characterization.
  • the molecular size variant determination method (CE-SDS) of the disclosed monoclonal antibody can adopt the sodium dodecyl sulfate capillary electrophoresis (CE-SDS) ultraviolet detection method, under reducing and non-reducing conditions, according to the molecular weight, according to the capillary electrophoresis method (2015 edition of "Chinese Pharmacopoeia” 0542), quantitatively determine the purity of recombinant monoclonal antibody products.
  • CE-SDS sodium dodecyl sulfate capillary electrophoresis
  • the glucocorticoid is coupled to the N-terminal amino group of the ligand and/or the ⁇ -amino group of the lysine residue through a linking unit.
  • the number of drug molecules will be less than the theoretical maximum.
  • the loading of antibody-drug conjugates can be controlled by the following non-limiting methods, including:
  • antibody refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds.
  • the amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different.
  • immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are ⁇ , ⁇ , and ⁇ chains, respectively. , ⁇ chain, and ⁇ chain.
  • IgG can be divided into different subclasses according to the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified as either kappa chains or lambda chains by difference in the constant region.
  • Each of the five Ig classes can have either a kappa chain or a lambda chain.
  • variable region The sequence of about 110 amino acids near the N-terminal of the antibody heavy chain and light chain varies greatly, which is the variable region (Fv region); the rest of the amino acid sequence near the C-terminal is relatively stable, which is the constant region.
  • the variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conserved sequences. Three hypervariable regions determine the specificity of antibodies, also known as complementarity determining regions (CDR).
  • CDR complementarity determining regions
  • Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of 3 CDR regions and 4 FR regions, and the sequence from the amino terminal to the carboxyl terminal is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • Antibodies of the present disclosure include murine antibodies, chimeric antibodies, humanized antibodies and fully human antibodies, preferably humanized antibodies and fully human antibodies.
  • murine antibody in this disclosure refers to an antibody prepared using a mouse according to the knowledge and skill in the art. In preparation, test subjects are injected with the specified antigen, and hybridomas expressing antibodies having the desired sequence or functional properties are isolated.
  • chimeric antibody is an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody.
  • To establish a chimeric antibody it is necessary to first establish a hybridoma that secretes a mouse-derived specific monoclonal antibody, then clone the variable region gene from the mouse hybridoma cell, and then clone the constant region gene of the human antibody as required, and then clone the mouse variable region gene It is connected with the human constant region gene to form a chimeric gene and inserted into an expression vector, and finally expresses the chimeric antibody molecule in a eukaryotic system or a prokaryotic system.
  • humanized antibody also known as CDR-grafted antibody (CDR-grafted antibody) refers to the antibody variable region framework grafted with mouse CDR sequences to humans, that is, different types of human germline antibodies Antibodies generated in the framework sequences. It can overcome the heterologous reaction induced by chimeric antibodies due to carrying a large amount of mouse protein components.
  • framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.
  • the germline DNA sequences of the 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), as well as in Kabat, EA et al.
  • the humanized antibody of the present disclosure also includes the humanized antibody after affinity maturation of CDR by phage display. Further descriptions of methods involving the use of mouse antibodies in humanization include, for example, Queen et al., Proc., Natl. 321, 522 (1986), Riechmann, et al., Nature, 332, 323-327 (1988), Verhoeyen, et al., Science, 239, 1534 (1988)].
  • the development of monoclonal antibodies has gone through four stages, namely: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully human monoclonal antibodies.
  • the present disclosure is a fully human monoclonal antibody.
  • the relevant technologies for the preparation of fully human antibodies mainly include: human hybridoma technology, EBV transformed B lymphocyte technology, phage display technology (phage display), transgenic mouse antibody preparation technology (transgenic mouse) and single B cell antibody preparation technology, etc.
  • antigen-binding fragment refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. It has been shown that fragments of full-length antibodies can be utilized to perform the antigen-binding function of the antibody.
  • binding fragments included in "antigen-binding fragments" include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F(ab') 2 fragments, comprising (iii) Fd fragment consisting of VH and CH1 domains; (iv) Fv fragment consisting of VH and VL domains of a single arm of an antibody; (v ) a single domain or dAb fragment (Ward et al., (1989) Nature 341:544-546) consisting of a VH domain; and (vi) isolated complementarity determining regions (CDRs) or (vii) optionally via A combination of two or more isolated CDRs joined by a synthetic linker.
  • CDRs complementarity
  • the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked by a synthetic linker using recombinant methods, thus making it possible to produce a single protein in which the VL and VH regions pair to form a monovalent molecule. chain (referred to as single-chain Fv (scFv); see, eg, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:5879-5883).
  • single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody.
  • Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.
  • Antibodies can be of different isotypes, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.
  • Fab is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity among fragments obtained by treating an IgG antibody molecule with the protease papain (cleaving the amino acid residue at position 224 of the H chain), in which About half and the entire L chain is held together by disulfide bonds.
  • F(ab')2 is an antibody having a molecular weight of about 100,000 and having antigen-binding activity and comprising two Fab regions connected at the hinge position obtained by digesting the lower portion of the two disulfide bonds in the IgG hinge region with the enzyme pepsin fragment.
  • Fab' is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity obtained by cleaving the disulfide bond in the hinge region of the above-mentioned F(ab')2.
  • the Fab' fragment can be produced by inserting DNA encoding a Fab' fragment of an antibody into a prokaryote expression vector or a eukaryote expression vector and introducing the vector into a prokaryote or eukaryote to express the Fab'.
  • single-chain antibody single-chain Fv or “scFv” is meant to comprise an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker molecules.
  • Such scFv molecules may have the general structure: NH2 -VL-linker-VH-COOH or NH2 -VH-linker-VL-COOH.
  • Suitable prior art linkers consist of the repeated GGGGS amino acid sequence or variants thereof, for example using 1-4 repeat variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448) .
  • linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur.J. Immuno 1.31:94-106, Hu et al. (1996) , Cancer Res. 56:3055-3061, Described by Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001 ), Cancer Immunol.
  • CDR refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contribute to antigen binding.
  • One of the most commonly used definitions of the six CDRs is provided by Kabat E.A. et al., (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242).
  • the Kabat definition of CDRs applies only to CDR1, CDR2, and CDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) of the light chain variable domain, and to CDR1, CDR L2, and L3 of the heavy chain variable domain.
  • CDR2 and CDR3 CDR H2, CDR H3 or H2, H3).
  • CDR1, HCDR2, HCDR3 there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region.
  • Amino acid sequence boundaries for CDRs can be determined using any of a variety of well-known schemes, including the "Kabat” numbering convention (see Kabat et al.
  • the CDR amino acid residues in the heavy chain variable domain are numbered 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3);
  • the CDR amino acid residues in the chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3).
  • the CDR amino acid numbers in VH are 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1), 50- 52 (LCDR2) and 91-96 (LCDR3).
  • the CDRs consist 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).
  • the numbering of CDR amino acid residues in VH is approximately 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3)
  • the numbering of CDR amino acid residues in VL is approximately 27-32 (CDR1 ), 50-52 (CDR2) and 89-97 (CDR3).
  • the CDR regions of antibodies can be determined using the program IMGT/DomainGap Align.
  • antibody framework refers to the portion of a variable domain VL or VH that serves as a scaffold for the antigen-binding loops (CDRs) of the variable domain. Essentially, it is a variable domain without CDRs.
  • Binding to IL-4R refers to being able to interact with human IL-4R (or its epitope or fragment).
  • the term "antigen-binding site” herein refers to a three-dimensional site recognized by an antibody or antigen-binding fragment herein.
  • epitope refers to the site on an antigen to which an immunoglobulin or antibody specifically binds.
  • An epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique spatial conformation (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Volume 66, G.E. Morris, Ed. (1996)).
  • antibodies bind with an affinity (KD) of less than about 10 "7M , eg, about less than 10 "8M , 10 "9M or 10 " 10M or less.
  • KD affinity
  • nucleic acid molecule refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if the promoter or enhancer affects the transcription of the coding sequence.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • the carrier is a "plasmid” which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • the vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • the vectors disclosed herein are capable of autonomous replication in the host cells into which they have been introduced (e.g., bacterial vectors and episomal mammalian vectors with a bacterial origin of replication) or can integrate into the genome of the host cell after introduction, thereby following The host genome is replicated together (eg, non-episomal mammalian vectors).
  • Antigen-binding fragments can also be prepared by conventional methods.
  • one or more human FR regions are added to the non-human CDR region by genetic engineering methods.
  • the human FR germline sequence can be obtained from the website http://imgt.cines.fr of ImMunoGeneTics (IMGT) by comparing the IMGT human antibody variable region germline gene database and MOE software, or from Immunoglobulin Journal, 2001ISBN012441351 get.
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells can include bacterial, microbial, plant or animal cells.
  • Bacteria that are readily transformed include members of the enterobacteriaceae such as strains of Escherichia coli or Salmonella; the 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 NSO cells.
  • Antibodies or antigen-binding fragments engineered in the present disclosure can be prepared and purified using conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian expression systems lead to glycosylation of antibodies, especially at the highly conserved N-terminal site of the Fc region. Positive clones are expanded in serum-free medium in bioreactors for antibody production.
  • the culture fluid from which the antibody has been secreted can be purified by conventional techniques. For example, use A or GSepharose FF columns with adjusted buffers for purification.
  • Antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and aggregates can also be removed by conventional methods such as molecular sieves and ion exchange. The obtained product needs to be immediately frozen, such as -70°C, or freeze-dried.
  • Amino acid sequence identity means that when the amino acid sequences are aligned and gaps are introduced as necessary to achieve the maximum percent sequence identity, and any conservative substitutions are not considered as part of the sequence identity, the difference between a first sequence and a second sequence is The percentage of amino acid residues that are identical to each other. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • anti-IL-4R antibody or "antibody that binds IL-4R” refers to an antibody that is capable of binding IL-4R, eg, with sufficient affinity such that the antibody is useful as a therapeutic agent targeting IL-4R.
  • the extent of binding of an anti-IL-4R antibody to an irrelevant non-IL-4R protein can be less than about 10% of the binding of the antibody to IL-4R as measured, for example, by radioimmunoassay (RIA).
  • the antibody that binds IL-4R has a dissociation constant (Kd) ⁇ 1 ⁇ M, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, or ⁇ 0.1 nM.
  • peptide refers to a compound fragment between amino acids and proteins, consisting of 2 or more amino acid molecules Peptide bonds are connected to each other and are structural and functional fragments of proteins, such as hormones and enzymes, which are essentially peptides.
  • sucrose refers to a biomacromolecule composed of three elements, C, H, and O, and can be divided into monosaccharides, disaccharides, and polysaccharides.
  • fluorescent probe refers to a characteristic fluorescence in the ultraviolet-visible-near-infrared region, and its fluorescence properties (excitation and emission wavelengths, intensity, lifetime and polarization, etc.) can vary with the properties of the environment, such as polarity, refractive index A class of fluorescent molecules that can be sensitively changed due to changes in viscosity, viscosity, etc., which can change one or several fluorescent properties by non-covalent interaction with nucleic acid (DNA or RNA), protein or other macromolecular structures, which can be used for research Properties and behavior of macromolecular substances.
  • DNA or RNA nucleic acid
  • protein or other macromolecular structures which can be used for research Properties and behavior of macromolecular substances.
  • alkyl refers to a saturated aliphatic hydrocarbon group, which is a linear or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 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-methylp
  • alkyl groups containing 1 to 6 carbon atoms 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-dimethyl Butyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl , 2,3-dimethylbutyl, etc.
  • Alkyl groups may be substituted or unsubstituted, and when substituted, substituents may be substituted at any available point of attachment, said substituents being preferably one or more of the following groups independently selected from alkyl radical, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkane Oxy group, heterocycloalkoxy group, cycloalkylthio group, heterocycloalkylthio group, oxo group, carboxyl group or carboxylate group.
  • heteroalkyl refers to an alkyl group containing one or more heteroatoms selected from N, O or S, wherein alkyl is as defined above.
  • cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring containing 3 to 20 carbon atoms, preferably containing 3 to 12 carbon atoms, more preferably containing 3 to 6 carbon atoms.
  • Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatriene Base, cyclooctyl, etc.; polycyclic cycloalkyl includes spiro ring, fused ring and bridged ring cycloalkyl. "Carbocycle” refers to the ring system in a cycloalkyl group.
  • spirocycloalkyl refers to a polycyclic group of 5 to 20 membered monocyclic rings sharing one carbon atom (called a spiro atom), which may contain one or more double bonds, but none of the rings has complete conjugation The ⁇ -electron system. Preferably it is 6 to 14 yuan, more preferably 7 to 10 yuan. According to the number of spiro atoms shared between the rings, the spirocycloalkyl group can be divided into single spirocycloalkyl, double spirocycloalkyl or polyspirocycloalkyl, preferably single spirocycloalkyl and double spirocycloalkyl.
  • spirocarbocycle refers to the ring system in a spirocycloalkyl.
  • Non-limiting examples of spirocycloalkyl Examples include:
  • fused cycloalkyl refers to a 5 to 20 membered all-carbon polycyclic group in which each ring of the system shares an adjacent pair of carbon atoms with other rings in the system, wherein one or more rings may contain one or Multiple double bonds, but none of the rings have a fully conjugated ⁇ -electron system. Preferably it is 6 to 14 yuan, more preferably 7 to 10 yuan. According to the number of rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic condensed cycloalkyl groups, preferably bicyclic or tricyclic, more preferably 5-membered/5-membered or 5-membered/6-membered bicycloalkyl groups.
  • fused carbocycle refers to the ring system in a fused cycloalkyl group. Non-limiting examples of fused cycloalkyl groups include:
  • bridged cycloalkyl refers to a 5 to 20 membered, all-carbon polycyclic group having any two rings sharing two carbon atoms not directly attached, which may contain one or more double bonds, but none of the rings has a complete Conjugated ⁇ -electron systems. Preferably it is 6 to 14 yuan, more preferably 7 to 10 yuan. According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic bridged cycloalkyl groups, preferably bicyclic, tricyclic or tetracyclic, more preferably bicyclic or tricyclic.
  • bridged cycloalkyl groups include:
  • the cycloalkyl ring may be fused to an aryl, heteroaryl or heterocycloalkyl ring where the ring bonded to the parent structure is a cycloalkyl, non-limiting examples include indanyl, tetrahydronaphthalene base, benzocycloheptyl, etc.
  • Cycloalkyl groups may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , heterocycloalkylthio, oxo, carboxyl or carboxylate.
  • heterocyclyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent comprising 3 to 20 ring atoms, one or more of which is selected from nitrogen, oxygen or S(O) m (where m is an integer from 0 to 2), but excluding ring portions of -OO-, -OS- or -SS-, the remaining ring atoms being carbon.
  • ring atoms Preferably it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably it contains 3 to 6 ring atoms.
  • Non-limiting examples of monocyclic heterocyclyl groups include pyrrolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, dihydroimidazolyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolyl, piperidine group, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc., preferably piperidinyl and pyrrolidinyl.
  • Polycyclic heterocyclyls include spiro, fused and bridged heterocyclyls. "Heterocycle" refers to a ring system in a heterocyclyl group.
  • spiroheterocyclyl refers to a polycyclic heterocyclic group that shares one atom (called a spiro atom) between 5 to 20-membered monocyclic rings, wherein one or more ring atoms are selected from nitrogen, oxygen or S(O ) m (wherein m is an integer from 0 to 2), the remaining ring atoms are carbon. It may contain one or more double bonds, but none of the rings has a fully conjugated pi-electron system. Preferably it is 6 to 14 yuan, more preferably 7 to 10 yuan.
  • the spiroheterocyclyl can be divided into single spiroheterocyclyl, double spiroheterocyclyl or polyspiroheterocyclyl, preferably single spiroheterocyclyl and double spiroheterocyclyl. More preferably, it is a 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered or 5-membered/6-membered monospiro heterocyclic group.
  • Spiroheterocycle refers to the ring system in a spiroheterocyclyl group.
  • Non-limiting examples of spiroheterocyclyls include:
  • fused heterocyclyl refers to a 5 to 20 membered polycyclic heterocyclic group in which each ring in the system shares an adjacent pair of atoms with other rings in the system, and one or more rings may contain one or more double bond, but none of the rings has a fully conjugated ⁇ -electron system, where one or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O) m (where m is an integer from 0 to 2), and the remaining ring
  • the atom is carbon.
  • it is 6 to 14 yuan, more preferably 7 to 10 yuan.
  • fused heterocyclic groups preferably bicyclic or tricyclic, more preferably 5-membered/5-membered or 5-membered/6-membered bicyclic fused heterocyclic groups.
  • fused heterocycle refers to a ring system within a fused heterocyclyl group.
  • fused heterocyclic groups include:
  • bridged heterocyclyl refers to a 5 to 14 membered polycyclic heterocyclic group in which any two rings share two atoms not directly attached, which may contain one or more double bonds, but none of the rings has a complete shared bond.
  • it is 6 to 14 yuan, more preferably 7 to 10 yuan.
  • bridged heterocyclyl groups include:
  • the heterocyclyl ring may be fused to an aryl, heteroaryl, or cycloalkyl ring, wherein the ring attached to the parent structure is heterocyclyl, non-limiting examples of which include: wait.
  • Heterocyclic groups may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alk Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , heterocycloalkylthio, oxo, carboxyl or carboxylate.
  • aryl refers to a 6 to 14 membered all-carbon monocyclic or fused polycyclic (that is, rings sharing adjacent pairs of carbon atoms) group, preferably 6 to 10 membered, having a conjugated pi-electron system, such as benzene base and naphthyl.
  • the aryl ring may be fused to a heteroaryl, heterocyclyl or cycloalkyl ring, where the ring bonded to the parent structure is the aryl ring.
  • “Aromatic ring” refers to a ring system in an aryl group. Non-limiting examples of aryl groups include:
  • Aryl groups may be substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, Alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycle Alkylthio, carboxyl or carboxylate, preferably phenyl.
  • fused ring aryl can be an unsaturated aromatic condensed ring structure containing 8-14 ring atoms connected by two or more ring structures sharing two adjacent atoms.
  • the number of atoms is preferably 8-12.
  • it includes all unsaturated condensed ring aryl groups, such as naphthalene, phenanthrene, etc., and also includes partially saturated condensed ring aryl groups, such as benzo 3-8 membered saturated monocyclic cycloalkyl, benzo 3-8 membered partially saturated monocyclic ring alkyl.
  • fused aromatic ring refers to a ring system in a fused aromatic group.
  • condensed ring aryl group examples include 2,3-dihydro-1H-indenyl, 1H-indenyl, 1,2,3,4-tetrahydronaphthyl, 1,4-dihydronaphthyl and the like.
  • heteroaryl refers to a heteroaromatic system comprising 1 to 4 heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen.
  • Heteroaryl is preferably 5 to 12 membered, such as imidazolyl, furyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, pyrrolyl, tetrazolyl, pyridyl, pyrimidinyl, thiadiazole, pyrazine group, etc., preferably imidazolyl, pyrazolyl, pyrimidinyl or thiazolyl; more preferably pyrazolyl or thiazolyl.
  • heteroaryl may be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring bonded to the parent structure is a heteroaryl ring.
  • Heteroaryl refers to a ring system in a heteroaryl group.
  • Non-limiting examples of heteroaryl groups include:
  • Heteroaryl groups may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , heterocycloalkylthio, carboxyl or carboxylate.
  • fused heteroaryl may contain 5-14 ring atoms (including at least one heteroatom), which is formed by connecting two or more ring structures with two adjacent atoms.
  • Aromatic condensed ring structure, including carbon atoms, nitrogen atoms and sulfur atoms can be oxo, preferably "5-12 membered fused heteroaryl", “7-12 membered fused heteroaryl”, “9-12 membered Fused heteroaryl” etc., such as benzofuryl, benzoisofuryl, benzothienyl, indolyl, isoindole, benzoxazolyl, benzimidazole, indazolyl, benzotri Azolyl, quinolinyl, 2-quinolinone, 4-quinolinone, 1-isoquinolinone, isoquinolinyl, acridinyl, phenanthridinyl, benzopyridazinyl, phthalazinyl, quinolinyl Azolinyl
  • Fused heteroaryl groups may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, Alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio group, heterocycloalkylthio group, carboxyl group or carboxylate group.
  • alkylene (-CH 2 -) refers to the remaining part of an alkane molecule after removing 2 hydrogen atoms, including straight-chain and branched-chain subgroups of 1 to 20 carbon atoms.
  • Alkylene groups containing 1 to 6 carbon atoms non-limiting examples include methylene (-CH 2 -), ethylene (such as -CH 2 CH 2 - or -CH(CH 3 )-), ethylene Propyl (eg -CH 2 CH 2 CH 2 - or -CH(CH 2 CH 3 )-), butylene (eg -CH 2 CH 2 CH 2 CH 2 -).
  • alkylene group may be substituted or unsubstituted, and when substituted, the substituent may be substituted at any available point of attachment, preferably one or more of the following groups independently selected from halogen, deuterium, hydroxyl, Nitro, cyano or amino.
  • alkenylene "heteroarylene”, “arylene”, “heterocycloalkylene” and the like are as defined above.
  • alkenylene refers to a group comprising linear alkenes having 2 to 8 carbon atoms, preferably 2 to 6 carbon atoms, more preferably 2 to 4 carbon atoms, and having at least one double bond in any position. groups, including, for example, vinylidene, allylene, propenylene, butenylene, prenylene, butadienylene, pentenylene, pentylene Alkenyl, hexenylene, hexadienylene, etc.
  • alkynylene includes linear alkynylene groups having 2 to 8 carbon atoms, preferably 2 to 6 carbon atoms, more preferably 2 to 4 carbon atoms, and having at least one triple bond in any position. groups, including, for example, ethynylene, propynylene, butynylene, pentynylene, hexynylene, and the like.
  • alkoxy refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl is as defined above.
  • alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy.
  • Alkoxy may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkoxy Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , heterocycle Alkylthio, carboxyl or carboxylate.
  • the substituent is preferably one or more of the following groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkoxy Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycl
  • alkylthio refers to -S-(alkyl) and -S-(unsubstituted cycloalkyl), wherein alkyl is as defined above.
  • alkylthio include: methylthio, ethylthio, propylthio, butylthio, cyclopropylthio, cyclobutylthio, cyclopentylthio, cyclohexylthio.
  • Alkylthio groups may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alk Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , One or more substituents in heterocycloalkylthio.
  • hydroxyalkyl refers to an alkyl group substituted by a hydroxy group, wherein alkyl is as defined above.
  • haloalkyl refers to an alkyl group substituted with a halogen, wherein alkyl is as defined above.
  • deuteroalkyl refers to an alkyl group substituted with a deuterium atom, wherein alkyl is as defined above.
  • hydroxyl refers to a -OH group.
  • a carbon atom is linked to an oxygen atom by a double bond, where a ketone or aldehyde group is formed.
  • a carbon atom is double bonded to a sulfur atom to form a thiocarbonyl group -C(S)-.
  • halogen refers to fluorine, chlorine, bromine or iodine.
  • amino refers to -NH2 .
  • cyano refers to -CN.
  • nitro refers to -NO2 .
  • aldehyde refers to -CHO.
  • carboxylate refers to -C(O)O(alkyl) or -C(O)O(cycloalkyl), wherein alkyl and cycloalkyl are as defined above.
  • acyl halide refers to a compound containing the group -C(O)-halogen.
  • sulfonyl refers to -S(O)(O)-.
  • amino-protecting group is a suitable group known in the art for amino-protection, referring to the amino-protecting group in the literature ("Protective Groups in Organic Synthesis", 5 Th.Ed.TW Greene & P.GMWuts), preferably , the amino protecting group can be (C 1-10 alkyl or aryl) acyl, for example: formyl, acetyl, benzoyl, etc.; can be (C 1-6 alkyl or C 6-10 aryl base) sulfonyl; it can also be (C 1-6 alkoxy or C 6-10 aryloxy) carbonyl, for example: Boc or Cbz; it can also be substituted or unsubstituted alkyl, for example: trityl 2,4-dimethoxybenzyl (DMB), p-methoxybenzyl (PMB) or benzyl (Bn).
  • DMB trityl 2,4-dimethoxybenzyl
  • PMB
  • Optional or “optionally” means that the subsequently described event or circumstance can but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur.
  • a heterocycloalkane group optionally substituted with an alkyl group means that an alkyl group may but need not be present, and the specification includes cases where the heterocycloalkane group is substituted with an alkyl group and where the heterocycloalkane group is not substituted with an alkyl group. situation of replacement.
  • pharmaceutical composition means a mixture containing one or more compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, as well as other components such as physiologically/pharmaceutically acceptable Carriers and Excipients.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, facilitate the absorption of the active ingredient and thus exert biological activity.
  • drug carrier used in the drug of the present disclosure refers to a system that can change the way the drug enters the human body and distributes the drug in the body, controls the release rate of the drug, and delivers the drug to the target organ.
  • Drug carrier release and targeting system enables reduction of drug Degradation and loss, reduce side effects, improve bioavailability.
  • polymer surfactants that can be used as carriers can self-assemble and form various forms of aggregates due to their unique amphiphilic structure.
  • Preferred examples are micelles, microemulsions, gels, liquid crystals, vesicles, etc. . These aggregates have the ability to entrap drug molecules, and at the same time have good permeability to the membrane, which can be used as excellent drug carriers.
  • excipient is an add-on in a pharmaceutical preparation other than the main drug, and can also be called an adjuvant.
  • adjuvant such as binders, fillers, disintegrants, lubricants in tablets; matrix parts in semi-solid ointments and creams; preservatives, antioxidants, flavoring agents, fragrances, Solubilizers, emulsifiers, solubilizers, osmotic pressure regulators, colorants, etc. can all be called excipients.
  • the term "diluent” is also known as filler and its main purpose is to increase the weight and volume of the tablet. The addition of the diluent not only ensures a certain volume, but also reduces the dose deviation of the main components and improves the compression molding properties of the drug. When the drug in the tablet contains oily components, an absorbent needs to be added to absorb the oily substance, so as to keep it in a "dry” state, so as to facilitate making tablets.
  • Compounds in the present disclosure may contain one or more asymmetric centers and thus may give rise to enantiomers, diastereoisomers, and which may be defined as (R)- or (S)-or according to absolute stereochemistry Other stereoisomeric forms of amino acids (D)- or (L)-.
  • This disclosure includes all possible isomers as well as their racemic and optically pure forms.
  • Optically active (+) and (-), (R)- and (S)- or (D)- and (L)-isomers can be prepared using chiral synthons or chiral reagents, or can be prepared using conventional methods Examples include chromatography and fractional crystallization.
  • the bond Indicates unassigned configuration, i.e. if chiral isomers exist in the chemical structure, the bond can be or or both and Two configurations.
  • the bond If the configuration is not specified, it can be Z configuration or E configuration, or both configurations.
  • Stepoisomer refers to compounds composed of the same atoms bonded by the same bonds but with different three-dimensional structures, which are not interchangeable.
  • Various stereoisomers and mixtures thereof are contemplated in this disclosure and include “enantiomers,” which refer to two stereoisomers whose molecules are non-superimposable mirror images of each other.
  • Tautomer refers to the transfer of a proton from one atom of a molecule to another atom of the same molecule. Tautomers of any of the described compounds are included in this disclosure.
  • Atoms capable of being isotopically labeled include, but are not limited to, hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, iodine, and the like. They can be replaced by isotopes 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, respectively.
  • deuterium when a position is specifically designated as deuterium (D), the position is understood to mean deuterium having an abundance of at least 3000 times greater than the natural abundance of deuterium (which is 0.015%) (ie, at least 45 % deuterium incorporation).
  • Figure 1 Dose-response curve of the whole-cell binding activity of the test protein to human IL-4R ⁇ -expressing cells
  • FIG. 4 Time-concentration curves of ADC-2 and free Budesonide in various tissues after intratracheal administration
  • NMR shifts ( ⁇ ) are given in units of 10 -6 (ppm).
  • the determination of NMR is to use Bruker AVANCE-400 nuclear magnetic instrument, and the determination solvent is deuterated dimethyl sulfoxide (DMSO-d 6 ), deuterated chloroform (CDCl 3 ), deuterated methanol (CD 3 OD), and the internal standard is four Methylsilane (TMS).
  • DMSO-d 6 dimethyl sulfoxide
  • CDCl 3 deuterated chloroform
  • CD 3 OD deuterated methanol
  • TMS Methylsilane
  • MS was determined by Shimadzu 2010 Mass Spectrometer or Agilent 6110AMSD mass spectrometer.
  • HPLC uses Shimadzu LC-20A systems, Shimadzu LC-2010HT series or Agilent Agilent 1200 LC high pressure liquid chromatography (Ultimate XB-C18 3.0*150mm column or Xtimate C18 2.1*30mm column).
  • Chiralpak IC-3 100 ⁇ 4.6mm I.D., 3um, Chiralpak AD-3 150 ⁇ 4.6mm I.D., 3um, Chiralpak AD-3 50 ⁇ 4.6mm I.D., 3um, Chiralpak AS-3 150 ⁇ 4.6mm were used for chiral HPLC analysis and determination I.D., 3um, Chiralpak AS-3 100 ⁇ 4.6mm I.D., 3 ⁇ m, ChiralCel OD-3 150 ⁇ 4.6mm I.D., 3um, Chiralcel OD-3 100 ⁇ 4.6mm I.D., 3 ⁇ m, ChiralCel OJ-H 150 ⁇ 4.6mm I.D., 5um, Chiralcel OJ-3 150 ⁇ 4.6mm I.D., 3um chromatographic column; use Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate for thin-layer chromatography silica gel plate, and the specification of silica gel plate used for thin-layer chromatography (TLC) is 0.15mm ⁇ 0.2mm, and the specifications
  • the chiral preparative column uses DAICEL CHIRALPAK IC (250mm*30mm, 10um) or Phenomenex-Amylose-1 (250mm*30mm, 5um).
  • the CombiFlash rapid preparation instrument uses Combiflash Rf150 (TELEDYNE ISCO).
  • the known starting materials of the present disclosure can be adopted or synthesized according to methods known in the art, or can be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Shaoyuan Chemical Technology (Accela ChemBio Inc), Darui Chemical products and other companies.
  • the reactions can all be carried out under an argon atmosphere or a nitrogen atmosphere.
  • Argon atmosphere or nitrogen atmosphere means that the reaction bottle is connected to an argon or nitrogen balloon with a volume of about 1 L.
  • the hydrogen atmosphere means that the reaction bottle is connected to a hydrogen balloon with a capacity of about 1L.
  • the pressurized hydrogenation reaction uses Parr 3916EKX hydrogenator and Qinglan QL-500 hydrogen generator or HC2-SS hydrogen Chemometer.
  • the hydrogenation reaction is usually vacuumized and filled with hydrogen, and the operation is repeated 3 times.
  • the solution refers to an aqueous solution.
  • reaction temperature is room temperature, which is 20°C to 30°C.
  • the monitoring of the reaction process in the embodiment adopts thin-layer chromatography (TLC), the developer used for reaction, the eluent system of the column chromatography that purifies compound adopts and the developer system of thin-layer chromatography comprise: A: Dichloromethane/methanol system, B: n-hexane/ethyl acetate system, C: petroleum ether/ethyl acetate system, D: petroleum ether/ethyl acetate/methanol, the volume ratio of the solvent is determined according to the polarity of the compound Adjustment can also be adjusted by adding a small amount of alkaline or acidic reagents such as triethylamine and acetic acid.
  • TLC thin-layer chromatography
  • DAST diethylaminosulfur trifluoride
  • THF tetrahydrofuran
  • NMP N-methylpyrrolidone
  • DCM dichloromethane
  • m-CPBA m-chloroperoxybenzoic acid
  • DIEA N,N-diisopropyl Ethylamine
  • TEA triethylamine
  • Boc tert-butoxycarbonyl, MeOH: methanol
  • Et 2 O ether.
  • Example 1 N-((10S)-10-benzyl-1-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxyl-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 , Preparation of 14-tetrapolyethylene glycol-16-yl)-1-(2-bromoacetamido)-3,6,9,12-tetraoxopentadecane-15-amide (compound 1)
  • Step 5 (9H-fluoren-9-yl)methyl(2-(((2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-di Methyl-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 base) preparation of carbamate (compound 1f)
  • Step 7) (9H-fluoren-9-yl)methyl((10S)-10-benzyl-1-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxyl-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)-1,6,9,12,15,18-hexaoxo Preparation of -3,21,24,27,30-pentoxo-5,8,11,14,17-pentaaminotriaconate-32-yl)carbamate (compound 1h)
  • Step 9) 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, Preparation of 14-tetrapolyethylene glycol-16-yl)-1-(2-bromoacetamido)-3,6,9,12-tetraoxopentadecane-15-amide (compound 1)
  • Step 2) (9H-fluoren-9-yl)methyl-((10S)-10-benzyl-1-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxyl- 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- Preparation of 3-oxo-5,8,11,14-tetrapolyethylene glycol-16-yl)carbamate (compound 2b)
  • the gene sequence of the synthetic antibody was subcloned into the pcDNA3.4 vector.
  • the ligation product was transformed into Top10 competent cells, and the positive clones were picked and expanded for culture.
  • the clones were inoculated in the culture medium to expand the culture, and a large number of plasmids containing the deoxyribose nucleic acid sequence of the 25G7-Fab antibody were extracted.
  • liposome transfection the expression plasmid was mixed with the transfection reagent, added to Expi 293 cells, cultured in a constant temperature incubator for 5 days, and the cell culture was harvested. After the cell culture was centrifuged, the supernatant was collected, the cell debris was removed by filtration, and the clarified liquid was collected.
  • AZD1402-TAG the protein sequence is from SEQ ID NO1 in patent WO2020200960A1, in order to facilitate protein Preparation and purification, the C-terminus of the sequence was fused with a His tag, and finally named AZD1402-TAG).
  • the coding gene sequence of the above protein was synthesized and subcloned into pcDNA3.4. After mixing the expression plasmid and the transfection reagent, incubate at 37°C for 15 minutes, add the mixture dropwise into the HEK293 cell fluid, and culture the cell fluid on a shaker at 37°C for one week, centrifuge the cell culture and take the supernatant. Use a buffer without imidazole to equilibrate the nickel affinity chromatography column, then pass the protein sample through the nickel affinity chromatography column for loading, and again equilibrate with the buffer without imidazole, then use the buffer containing high concentration of imidazole Elute column-bound protein. The eluted protein was transferred to a dialysis bag, dialyzed in 1 ⁇ PBS, and replaced with PBS storage buffer. After detection, the target protein was obtained.
  • Buffer A In a 2.0L container, add KH 2 PO 4 (8.50g), K 2 HPO 4 (8.56g), NaCl (5.86g) and EDTA (1.50g), add 1.6L water for injection, stir Half an hour, after completely dissolving, dilute to 2.0L with water for injection, and measure the pH to be 6.30 ⁇ 0.1.
  • TCEP tris(2-carboxyethyl)phosphine
  • TCEP tris(2-carboxyethyl)phosphine
  • Test Example 1 Affinity detection between antibody drug conjugate ADC-2 and human IL-4R ⁇ protein
  • the flow rate was set at 30 ⁇ L/min.
  • the detection time of ADC-2, 25G7-Fab and AZD1402-TAG was 100 seconds for binding and 100 seconds for dissociation. 600 seconds.
  • Biacore 8K evaluation software (Cytiva) was used for analysis to obtain the affinity data of each test protein.
  • Test Example 2 Whole-cell Binding Activity of Antibody Drug Conjugate ADC-2 to Human IL-4R ⁇ Expressing Cells
  • Antibody drug conjugates ADC-2, 25G7-Fab, hu25G7 (heavy chain sequence shown in SEQ ID NO: 44, light chain sequence shown in SEQ ID NO: 45), AZD1402-TAG.
  • TF-1 and Karpas 299 cell lines were identified to express IL-4R ⁇ . After the TF-1 and Karpas299 cells in the logarithmic growth phase were collected, the cell density was counted and adjusted, and placed in a round-bottom 96-well plate at 1 ⁇ 10 5 cells/well. Cells were incubated with different concentrations of anti-IL-4R antibodies (25G7, naked anti-25G7-Fab), antibody-drug conjugates ADC-2 and AZD1402-TAG at 4°C for 45 minutes, and FACS Buffer (containing 2% FBS After washing away the antibody, fluorescently labeled secondary antibody was added for staining.
  • anti-IL-4R antibodies 25G7, naked anti-25G7-Fab
  • ADC-2 and AZD1402-TAG antibody-drug conjugates ADC-2 and AZD1402-TAG
  • 25G7-Fab, hu25G7 and ADC-2 were stained with FITC-coupled anti-human IgG (Fab specific) secondary antibody (Sigma, CAT#F5512-1ML), AZD1402-TAG uses FITC-conjugated anti-His antibody (GenScript, CAT#A01620).
  • the cells were incubated with the secondary antibody at 4°C for 30 minutes, then washed twice with FACS Buffer, detected by flow cytometry (BD, FACS Celesta), and the detection results were analyzed by FlowJo (FlowJo, LLC) software.
  • Table 2 The EC50 value of the whole cell binding activity of the test protein to human IL-4R ⁇ expressing cells
  • Test Example 3 Blocking Effect of Antibody Drug Conjugate ADC-2 on IL-4/IL-13 Signaling Pathway
  • the activation of STAT6 is a key step in the activation of IL-4/IL-13 signaling pathway.
  • the blocking effect of ADC-2 on IL-4/IL-13 signaling pathway was evaluated by HEK-Blue TM IL-4/IL-13 reporter gene cell line.
  • the cells were purchased from Invivogen (Cat#hkb-il413), in which the human STAT6 gene and the secreted alkaline phosphatase reporter gene (Secreted alkaline phos-phatase, SEAP) induced by phosphorylated STAT6 were overexpressed.
  • SEAP substrate QUANTI-Blue was used to detect the secreted SEAP content in the cell culture supernatant to assess the activation level of the IL-4/IL-13 signaling pathway.
  • each of the tested proteins can block the activation of STAT6 induced by recombinant human IL-4 and IL-13, and the IC50 value of ADC-2 for blocking the IL-4 signaling pathway is 0.68nM, which is close to the IC 50 value of 0.61nM of 25G7-Fab; the IC 50 value of ADC-2 blocking IL-13 signaling pathway is 10.60nM, which is comparable to the IC 50 value of 25G7-Fab of 14.59nM.
  • Test Example 4 Endocytic Activity of Antibody Drug Conjugate ADC-2 in TF-1 Cells
  • Antibody drug conjugates ADC-2, 25G7-Fab, hu25G7, AZD1402-TAG.
  • Collect well-growing TF-1 cells and adjust the density to 1 ⁇ 10 6 cells/mL add 100 ⁇ L/well into a 96-well cell culture plate and place at 4°C, add each test protein at a final concentration of 2 nM, and After incubating at 4°C for 1 hour, put it into the cell culture incubator at 4°C for 1 minute, and continue to cultivate under the condition of 5% CO 2 , set up a culture plate at each time point, take out the culture plate according to different incubation times, wash with FACS buffer and centrifuge , followed by incubation with fluorescently-conjugated secondary antibodies.
  • 25G7-Fab, hu25G7 and ADC-2 were labeled with FITC-conjugated anti-human IgG (Fab-specific) secondary antibody, and AZD1402-TAG was labeled with FITC-conjugated anti-His antibody.
  • the cells were incubated with the secondary antibody at 4°C for 30 minutes, then washed twice with FACS Buffer, detected by flow cytometry (BD, FACS Celesta), and the detection results were analyzed by FlowJo (FlowJo, LLC) software.
  • the formula for calculating the endocytosis rate at a specific time point is: (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%
  • Test Example 5 Pharmacokinetic test of antibody drug conjugate ADC-2 in mouse model
  • mice received intratracheal atomized administration (intratracheal, i.t.), and 300-400 ⁇ L of jaw blood was collected at different time points after administration, and after standing for 2 hours, centrifuged at 7500 rpm and 4°C for 10 min, serum was collected. After blood collection, the mice were anesthetized and fixed, and the neck muscles of the mice were bluntly dissected to expose the trachea. The trachea was opened laterally, and the lavage needle was slowly inserted, and the pre-pumped 4°C pre-cooled saline was slowly pushed in. Lung tissue was lavaged, and alveolar lavage fluid (BALF) was collected.
  • intratracheal intratracheal, i.t.
  • Blocking add 300 ⁇ L of PBS containing 3% BSA to each well, shake the plate at room temperature at 400 rpm and incubate for 2 hours.
  • the detection antibody (Invitrogen, A18811) was diluted 10,000 times with Assay buffer (0.05% PBST containing 0.5% BSA), added to the ELISA plate, 100 ⁇ L per well, and incubated for 1 h at room temperature at 400 rpm.
  • Plate washing add 300 ⁇ L 0.05% PBST to each well, and wash the plate 3 times.
  • Termination 100 ⁇ L of substrate reaction termination solution (Solarbio, C1058) was added to each well, and the light absorption value at a wavelength of 620 nm was detected.
  • the pharmacokinetic parameters of the antibody-drug conjugate ADC-2 and free Budesonide are shown in Table 6, and the drug-time curves are shown in FIG. 4 .
  • Test Example 6 In vivo efficacy of ADC-2 on OVA-induced mouse asthma model
  • mice were randomly divided into normal control animals and model animals. On Day 0, 7 and 14, 100 ⁇ L of PBS solution containing 200 ⁇ g OVA (Sigma, A5503) was injected intraperitoneally, and mice were sensitized with an equal volume of aluminum hydroxide as an adjuvant. On Day 21-27, the mice inhaled the nebulized solution of 3% OVA once a day for 35 minutes each time. Beginning on Day 21, half an hour before inhaling the OVA nebulized solution, the mice received intratracheal nebulized administration. The specific dosing regimen is shown in Table 7.
  • the number of white blood cells and typed cells in BALF The number of white blood cells and typed cells in BALF.
  • the experimental results are shown in Figure 5.
  • the antibody drug conjugate ADC-2 significantly reduced the number of white blood cells, eosinophils and neutrophils in the BALF, and also had a certain effect on the number of monocytes.
  • the efficacy is better than AZD1402-TAG, and significantly better than 25G7-Fab and Budesonide.
  • ADC-2 shows the synergistic effect of 25G7-Fab and Budesonide in terms of drug efficacy.
  • Test Example 7 Dose exploration of ADC-2 in vivo efficacy in OVA-induced mouse asthma model
  • the administration and BALF sampling operations are the same as in Test Example 6.
  • the specific dosage regimen is shown in Table 8.
  • the number of white blood cells and typed cells in BALF The number of white blood cells and typed cells in BALF.

Abstract

式（I）所示的抗体-药物偶联物，其中，Ab为抗体或其抗原结合片段， L为将Ab共价连接于D的连接子，且k为1至20， D为糖皮质激素或其残基，所述糖皮质激素选自布地奈德或环索奈德。 Ab-(L-D)k （I）

Description

糖皮质激素的药物偶联物 技术领域
本公开属于医药领域，具体涉及一种糖皮质激素的药物偶联物。
背景技术
白介素4(Interleukin-4，IL-4)由153个氨基酸组成，分子量约为17kDa。最初，因为IL-4能够刺激B细胞增殖而被发现，并被命名为B细胞刺激因子-1(BSF-1)。IL-4与IL-13一样属于I型细胞因子家族，具有4α螺旋的疏水束核心所构成的四级结构。IL-4由TH2细胞分泌，参与TH2介导的免疫应答，具有广泛的生物学活性，包括刺激T细胞、肥大细胞、粒细胞、巨核细胞和红细胞增殖。此外，IL-4还可以刺激B细胞表达主要组织相容性复合物2类分子。IL-13与IL-4具有大约30％的氨基酸序列同源性和多种相似的功能。IL-4和IL-13都能促进B细胞增殖，并联合CD40/CD40L共刺激诱导IgM类型转变成IgE。IL-4促进肥大细胞聚集，上调肥大细胞高亲和力IgE受体和B细胞上IgE低亲和力受体CD23(FcεRII)的表达，上调血管内皮细胞黏附分子(VCAM-1)表达，促进嗜酸性粒细胞、T淋巴细胞、单核细胞和嗜碱性粒细胞的转移。与IL-13不同的是，IL-4可以促进幼稚T细胞分化成TH2。
IL-4需要与膜受体结合而发挥生物学功能。人白介素受体(IL-4R)是由两条多肽链形成的异二聚体，其中一条α链对IL-4有很高的亲和力，由于在IL-4R复合物中IL-4Rα链对IL-4的结合起主导作用，因此很多科学研究和报道中常用IL-4Rα替代IL-4R。IL-4R在人B细胞、肥大细胞、嗜酸性粒细胞、嗜碱性粒细胞、巨噬细胞/单核细胞、DC细胞、纤维细胞、气道上皮和平滑肌等多种细胞上都有表达。IL-4Rα可以与其他亚基形成两类受体复合物，在造血干细胞中主要表达由IL-4Rα和γc组成的I型受体。在非造血干细胞中IL-4主要通过IL-4Rα和IL-13Rα1组成的II型受体发挥作用。II型受体是IL-4和IL-13的共同受体，IL-13与IL-13Rα1结合发挥功能。I型受体和II型受体都通过Jak/STAT通路转导信号，IL-4Rα、γc和IL-13Rα1分别与Jak1、Jak3和Tyk2结合激活下游通路，IL-4和IL-13还可以通过胰岛素受体底物家族(IRS)转导信号，最终激活核内的PI3-K、NF-κB。阻断IL-4R既可以抑制IL-4也可以抑制IL-13的生物学功能。
多项研究表明IL-4和IL-13与TH2免疫应答相关的疾病有关。特应性皮炎(AD)，又称异位性皮炎或遗传过敏性皮炎，是皮肤科常见疾病，多见于儿童和青少年，常与某些遗传过敏性疾病如过敏性鼻炎、哮喘等并发。研究发现AD患者的TH2因子IL-4、IL-5、IL-10和IL-13水平上升，IgE水平升高，此外还发现，TH2因子与AD疾病进程相关，过表达IL-4、IL-13等TH2因子的小鼠表现出皮肤保护缺陷和类似AD病症[14][15]。AD患者IL-4和IL-13水平升高阻碍了表皮分化和抗菌肽的产生。IL-4缺陷小鼠降低皮肤过敏性炎症的发生。这些研究表明阻断IL-4R可能对治疗AD有效。国外已经有抗IL-4R的单抗上市，对AD表现出良好的治疗效果。
IL-13和IL-4在哮喘中也发挥重要作用。哮喘是一种常见的肺部炎症疾病，以气道高反应性(AHR)、粘液分泌过多、纤维化和IgE水平升高为特征。非特异性刺激如冷空气等常常 导致气道高反应性加剧，AHR和粘液分泌过多导致气道阻碍这是哮喘致死的主要原因。TH2因子在哮喘疾病进程中发挥重要作用，哮喘患者支气管和肺泡灌洗液过表达IL-4和IL-13。尽管IL-13和IL-4具有某些功能相似性，但是一些研究表明IL-13在哮喘的疾病进展中发挥了比其他Th2细胞因子更为重要的作用。IL-13可以促进杯状细胞的分化和纤维化。给未经过过敏原刺激的小鼠气道注射重组IL-13会导致气道炎症，粘液分泌过多和气道高反应性，注射可溶性的IL13Rα2可以阻止小鼠AHR、粘液分泌过多和肺部炎症的发生。在哮喘模型中注射IL-4Rα抗体可以降低AHR和肺泡灌洗液中的嗜酸性粒细胞。研究表明阻断IL-4Rα可能对治疗哮喘有效。
目前各国已有多家制药公司正在研发针对IL-4R的单克隆抗体，相关专利申请如WO2010053751、WO2001092340、WO2008054606、WO2014031610、WO2020038454等。
糖皮质激素也是治疗过敏性疾病、炎症等较为有效的药物。作为代表性的糖皮质激素，已知皮质醇、皮质酮等在生物体内制成的糖皮质激素以及地塞米松、泼尼松、泼尼松龙、布地奈德等合成糖皮质激素。这些糖皮质激素由于具有类固醇结构，因此总称为类固醇类，应用在各种疾病的治疗中。但是，这些类固醇类由于其使用，有时会表现出类固醇消化性溃疡、类固醇紫斑、类固醇胰炎、类固醇糖尿病、类固醇白内障、类固醇青光眼等副作用。
抗体药物偶联物(antibody drug conjugate，ADC)，是指单克隆抗体或者抗体片段通过稳定的化学接头化合物与具有生物活性的药物相连。临床前和临床开发中的大多数ADC都用于肿瘤适应症，其中细胞毒性有效载荷靶向表达抗原的癌细胞。但是，通过ADC介导的生物活性小分子的传递来调节病原性细胞活性对于非肿瘤学适应症也是有吸引力的，从而导致了该技术的广泛应用。
现有技术已经公开了一些糖皮质激素的药物偶联物，例如WO2017210471，WO2019106609、WO2019136487等。
发明内容
本公开一方面提供了一种式(I)所示的抗体-药物偶联物(ADC)，
Ab-(L-D)k
(I)
其中，Ab为抗IL-4R的抗体或其抗原结合片段，
L为将Ab共价连接于D的连接子，且k为1至20(包括1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或任意两数值之间任意数值)，
D为糖皮质激素或其残基，所述糖皮质激素选自地塞米松、泼尼松、泼尼松龙、布地奈德、莫米松、丙酸倍氯米松、氟替卡松、曲安奈德和环索奈德，例如可以是布地奈德或环索奈德。
在一些实施方案中，D如下式所示：
在一些实施方案中，抗IL-4R的抗体或其抗原结合片段可以是已知的，如在例如WO2010053751、WO2001092340、WO2008054606、WO2014031610、WO2020038454(其各自通过引用并入本文)中所描述的抗IL-4R的抗体或其抗原结合片段。
在一些实施方案中，抗IL-4R的抗体或其抗原结合片段包括但不限于Dupixent、PRS-060、AK-120、63 IgG1、CBP201、AMG-317或其抗原结合片段。
在一些实施方案中，抗IL-4R的抗体或其抗原结合片段是抗人IL-4R的抗体或其抗原结合片段。
在一些实施方案中，抗IL-4R的抗体或其抗原结合片段包含重链可变区和轻链可变区，其中重链可变区包含：
(I)分别如SEQ ID NO：3、SEQ ID NO：4和SEQ ID NO：5所示的HCDR1、HCDR2和HCDR3；或
(II)分别如SEQ ID NO：11、SEQ ID NO：12和SEQ ID NO：13所示的HCDR1、HCDR2和HCDR3；
和/或抗体轻链可变区包含：
(I)分别如SEQ ID NO：6、SEQ ID NO：7和SEQ ID NO：8所示的LCDR1、LCDR2和LCDR3；或
(II)分别如SEQ ID NO：14、SEQ ID NO：15和SEQ ID NO：16所示的LCDR1、LCDR2和LCDR3；或
(III)分别如SEQ ID NO：38、SEQ ID NO：7和SEQ ID NO：40所示的LCDR1、LCDR2和LCDR3；或
(IV)分别如SEQ ID NO：42、SEQ ID NO：39和SEQ ID NO：8所示的LCDR1、LCDR2和LCDR3。
表1示出了抗IL-4R的抗体或其抗原结合片段的CDR序列。
表1抗IL-4R的抗体或其抗原结合片段的CDR序列

在一些实施方案中，抗IL-4R的抗体或其抗原结合片段包含选自以下(I)至(IV)中的任一项：
(I)重链可变区，其包含分别如SEQ ID NO：3、SEQ ID NO：4和SEQ ID NO：5所示的HCDR1、HCDR2和HCDR3；和
轻链可变区，其包含分别如SEQ ID NO：6、SEQ ID NO：7和SEQ ID NO：8所示的LCDR1、LCDR2和LCDR3；
(II)重链可变区，其包含分别如SEQ ID NO：11、SEQ ID NO：12和SEQ ID NO：13所示的HCDR1、HCDR2和HCDR3；和
轻链可变区，其包含分别如SEQ ID NO：14、SEQ ID NO：15和SEQ ID NO：16所示的LCDR1、LCDR2和LCDR3；
(III)重链可变区，其包含分别如SEQ ID NO：3、SEQ ID NO：4和SEQ ID NO：5所示的HCDR1、HCDR2和HCDR3；和
轻链可变区，其包含分别如SEQ ID NO：38、SEQ ID NO：7和SEQ ID NO：40所示 的LCDR1、LCDR2和LCDR3；
(IV)重链可变区，其包含分别如SEQ ID NO：3、SEQ ID NO：4和SEQ ID NO：5所示的HCDR1、HCDR2和HCDR3；和
轻链可变区，其包含分别如SEQ ID NO：42、SEQ ID NO：39和SEQ ID NO：8所示的LCDR1、LCDR2和LCDR3。
在一些实施方案中，所述的抗IL-4R的抗体或抗原结合片段是鼠源抗体、嵌合抗体、全人抗体、人源化抗体或其片段。在一些具体的实施方案中，所述抗IL-4R的抗体或抗原结合片段是人源化的。
在一些实施方案中，所述的抗IL-4R的抗体或其抗原结合片段，包含来源于人种系轻链IGKV3-11*01(SEQ ID NO：22，用于抗体25G7)的FR序列或与其至少有95％同一性的带有回复突变的序列。在一些具体实施方案中，所述的回复突变选自L46P，L47W，F71Y中的一个或多个。在一些实施方案中，所述的抗IL-4R的抗体或其抗原结合片段，包含来源于人种系重链IGHV3-48*01(SEQ ID NO：21，用于抗体25G7)的FR区序列或与其至少有95％同一性的带有回复突变的序列。在一些具体实施方案中，所述的回复突变选自S94A，F67S，A93T中的一个或多个。在一些实施方案中，所述的抗IL-4R的抗体或其抗原结合片段，包含来源于人种系轻链IGKV2D-29*01(SEQ ID NO：24，用于抗体7B10)的FR区序列或与其至少有95％同一性的回复突变序列。在一些具体实施方案中，所述的回复突变选自M4L和/或V58I。在一些实施方案中，所述的抗IL-4R的抗体或其抗原结合片段，包含来源于人种系重链IGHV1-2*02(SEQ ID NO：23，用于抗体7B10)的FR区序列或与其至少有95％同一性的回复突变序列。在一些具体实施方案中，所述的回复突变选自M69L，R71I，T73K，R94K中的一个或多个。
人种系重链IGHV3-48*01：
人种系轻链IGKV3-11*01：
人种系重链IGHV1-2*02：
人种系轻链IGKV2D-29*01：

在一些实施方案中，抗IL-4R的抗体或其抗原结合片段包含重链可变区和轻链可变区，其中：
重链可变区包含：
(I)如SEQ ID NO：1所示或与SEQ ID NO：1具有至少70％、80％、90％、95％、98％、99％同一性的序列；或
(II)如SEQ ID NO：9所示或与SEQ ID NO：9具有至少70％、80％、90％、95％、98％、99％同一性的序列；或
(III)如SEQ ID NO：43所示或与SEQ ID NO：43具有至少70％、80％、90％、95％、98％、99％同一性的序列；或
和/或轻链可变区包含：
(I)如SEQ ID NO：2所示或与SEQ ID NO：2具有至少70％、80％、90％、95％、98％、99％同一性的序列；或
(II)如SEQ ID NO：10所示或与SEQ ID NO：10具有至少70％、80％、90％、95％、98％、99％同一性的序列；或
(III)如SEQ ID NO：37所示或与SEQ ID NO：37具有至少70％、80％、90％、95％、98％、99％同一性的序列；或
(IV)如SEQ ID NO：41所示或与SEQ ID NO：41具有至少70％、80％、90％、95％、98％、99％同一性的序列。
25G7 HCVR(25G7重链可变区)
25G7 LCVR(25G7轻链可变区)
7B10 HCVR(7B10重链可变区)
7B10 LCVR(7B10轻链可变区)
hu25G7-ALCVR(hu25G7-A轻链可变区)
hu25G7-B LCVR(hu25G7-B轻链可变区)
hu25G7-VH(hu25G7重链可变区)
在至少一个实施方案中，所述抗IL-4R的抗体或抗原结合片段的重链可变区如序列SEQ ID NO：1所示，轻链可变区如序列SEQ ID NO：2所示；或
重链可变区如序列SEQ ID NO：9所示，轻链可变区如序列SEQ ID NO：10所示；或
重链可变区如序列SEQ ID NO：43所示，轻链可变区如序列SEQ ID NO：37所示；或
重链可变区如序列SEQ ID NO：43所示，轻链可变区如序列SEQ ID NO：41所示。
在一些实施方案中，抗IL-4R的抗体或其抗原结合片段包含重链可变区和轻链可变区，其中：
重链可变区包含：
(I)如SEQ ID NO：25-27之一所示或与SEQ ID NO：25-27之一具有至少70％、80％、90％、95％、98％或99％同一性的序列；或
(II)如SEQ ID NO：31-33之一所示或与SEQ ID NO：31-33之一具有至少70％、80％、90％、95％、98％、或99％同一性的序列；
和/或轻链可变区包含：
(I)如SEQ ID NO：28-30之一所示或与SEQ ID NO：28-30之一具有至少70％、80％、90％、95％、98％或99％同一性的序列；或
(II)如SEQ ID NO：34-36之一所示或与SEQ ID NO：34-36之一具有至少70％、80％、90％、95％、98％或99％同一性的序列。
hu25G7-VH-a：
hu25G7-VH-b：

hu25G7-VH-c：
hu25G7-VL-a：
hu25G7-VL-b：
hu25G7-VL-c：
hu7B10-VH-a：
hu7B10-VH-b：
hu7B10-VH-c：
hu7B10-VL-a：
hu7B10-VL-b：
hu7B10-VL-c：
在一些具体的实施方案中，重链可变区如序列SEQ ID NO：25-27之一所示，和轻链可变区如序列SEQ ID NO：28-30之一所示；
重链可变区如序列SEQ ID NO：31-33之一所示，和轻链可变区如序列SEQ ID NO：34-36之一所示。
在一些实施方案中，所述的抗IL-4R的抗体或其抗原结合片段包含选自人源IgG1、IgG2、IgG3或IgG4的重链恒定区或其变体。一些具体的方案中，包含人源IgG1的重链恒定区或其变体。在一些实施方案中，所述的抗IL-4R的抗体或其抗原结合片段包含人源κ、λ链或其变体的恒定区。
在一些实施方案中，所述的抗IL-4R的抗体或其抗原结合片段，其中所述抗体为人源化抗体，重链序列如SEQ ID NO：17所示或与其具有至少85％序列同一性；轻链序列如SEQ ID NO：18所示或与其具有至少85％序列同一性。
在一些实施方案中，所述的抗IL-4R的抗体或其抗原结合片段，其中所述抗体为人源化抗体，重链序列如SEQ ID NO：19所示或与其具有至少85％序列同一性；轻链序列如SEQ ID NO：20所示或与其具有至少85％序列同一性。
在一些实施方案中，所述的抗IL-4R的抗体或其抗原结合片段，其中所述抗体为人源化抗体，重链序列如SEQ ID NO：44所示或与其具有至少85％序列同一性；轻链序列如SEQ ID NO：45所示或与其具有至少85％序列同一性。
在一些实施方案中，所述的抗IL-4R的抗体或其抗原结合片段，其中所述抗体为人源化抗体，重链序列如SEQ ID NO：44所示或与其具有至少85％序列同一性；轻链序列如SEQ ID NO：46所示或与其具有至少85％序列同一性。
在一些实施方案中，提供一种分离的抗IL-4R抗体或其抗原结合片段，其特征在于与如上所述的任一抗IL-4R的抗体或其抗原结合片段竞争结合人IL-4R或其表位。
在一些实施方案中，提供一种双特异性抗体或多特异性抗体，其含有如上所述任一抗IL-4R的抗体或其抗原结合片段轻链可变区和/或重链可变区。在另一些实施方案中，提供一种单链抗体，其含有如上所述任一抗IL-4R的抗体或其抗原结合片段的轻链可变区和/或重链可变区。
在一些实施方案中，人源化抗IL-4R抗体或其抗原结合片段，还包含人源IgG1、IgG2、IgG3或IgG4的重链恒定区或其变体。在至少一个实施方案中，包含的是人源IgG2或IgG4重链恒定区。因为IgG2或IgG4没有ADCC毒性。在另一个实施方案中，使用氨基酸突变后无ADCC(antibody-dependent cell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用)毒性的IgG1。在至少一个实施方案中，变体包含ADCC效应功能降低或缺失的重链恒定区突变，例如但不限于IgG1的N297A、L234A、L235A。在一些实施方案中，IgG1包含突变E239D和M241L。
除非另有说明，本公开的抗IL-4R抗体或其抗原结合蛋白根据Kabat编码。
在一些实施方案中，抗IL-4R的抗体或其抗原结合片段的包含重链和轻链，其中重链包含：
(I)如SEQ ID NO：17所示或与SEQ ID NO：17具有至少70％、80％、90％、95％、98％或99％同一性的序列；或
(II)如SEQ ID NO：19所示或与SEQ ID NO：19具有至少70％、80％、90％、95％、98％、或99％同一性的序列；或
(III)如SEQ ID NO：44所示或与SEQ ID NO：44具有至少70％、80％、90％、95％、98％、或99％同一性的序列；
(IV如SEQ ID NO：47所示或与SEQ ID NO：47具有至少70％、80％、90％、95％、98％、或99％同一性的序列；
和/或轻链包含：
(I)如SEQ ID NO：18所示或与SEQ ID NO：18具有至少70％、80％、90％、95％、98％或99％同一性的序列；或
(II)如SEQ ID NO：20所示或与SEQ ID NO：20具有至少70％、80％、90％、95％、98％或99％同一性的序列；或
(III)如SEQ ID NO：45所示或与SEQ ID NO：45具有至少90％、95％、98％或99％同一性的序列；或
(IV)如SEQ ID NO：46所示或与SEQ ID NO：46具有至少90％、95％、98％或99％同一性的序列。
hu25G7 HC
hu25G7 LC
hu7B10 HC
hu7B10 LC
hu25G7-IgG4 HC
hu25G7-ALC
hu25G7-B LC
hu25G7-24-IgG1(E239D，M241L)HC
在一些实施方案中，重链序列如SEQ ID NO：17所示，和轻链序列如SEQ ID NO：18所示；或
重链序列如SEQ ID NO：19所示，和轻链序列如SEQ ID NO：20所示；或
重链序列如SEQ ID NO：44所示，和轻链序列如SEQ ID NO：45所示；或
重链序列如SEQ ID NO：44所示，和轻链序列如SEQ ID NO：46所示；或
重链序列如SEQ ID NO：47所示，和轻链序列如SEQ ID NO：45所示。
在一些实施方案中，抗IL-4R的抗体或其抗原结合片段的包含重链和轻链，其中重链包含：
(I)如SEQ ID NO：48所示或与SEQ ID NO：48具有至少70％、80％、90％、95％、98％或99％同一性的序列；或
(II)如SEQ ID NO：49所示或与SEQ ID NO：49具有至少70％、80％、90％、95％、98％、或99％同一性的序列；或
(III)如SEQ ID NO：50所示或与SEQ ID NO：50具有至少70％、80％、90％、95％、98％、或99％同一性的序列；
(IV如SEQ ID NO：51所示或与SEQ ID NO：51具有至少70％、80％、90％、95％、98％、或99％同一性的序列；
和/或轻链包含：
(I)如SEQ ID NO：18所示或与SEQ ID NO：18具有至少70％、80％、90％、95％、98％或99％同一性的序列；或
(II)如SEQ ID NO：45所示或与SEQ ID NO：45具有至少90％、95％、98％或99％同一性的序列；或
(III)如SEQ ID NO：46所示或与SEQ ID NO：46具有至少90％、95％、98％或99％同一性的序列。
hu25G7-IgG4 VH-CH1

hu25G7-IgG1 VH-CH1
hu25G7-24-IgG4 VH-CH1
hu25G7-24-IgG1 VH-CH1
在一些实施方案中，重链序列如SEQ ID NO：48所示，和轻链序列如SEQ ID NO：18所示；或
重链序列如SEQ ID NO：49所示，和轻链序列如SEQ ID NO：18所示；或
重链序列如SEQ ID NO：50所示，和轻链序列如SEQ ID NO：45所示；或
重链序列如SEQ ID NO：50所示，和轻链序列如SEQ ID NO：46所示；或
重链序列如SEQ ID NO：51所示，和轻链序列如SEQ ID NO：45所示；或
重链序列如SEQ ID NO：51所示，和轻链序列如SEQ ID NO：46所示。
在一些实施方案中，所述抗IL-4R抗体或其抗原结合片段可以为抗体变体，所述变体在轻链有1至10个(例如1、2、3、4、5、6、7、8、9、10个)氨基酸变化，和/或重链有1至10个(例如1、2、3、4、5、6、7、8、9、10个)氨基酸变化。
在一些实施方案中，上述变体具有与亲本抗IL-4R的抗体或其片段相同或相似的生物学功能或效果。
在一些实施方案中，抗原结合片段包括但不限于Fab、Fab’、Fv、F(ab’)2、线性抗体、scFv(单链Fv抗体)、串联二-scFv、串联三-scFv、双链抗体(diabody)、三链抗体(triabody)、 四链抗体(tetrabody)、sdAb(单域抗体或纳米抗体)、sdFv、肽抗体(peptibody)、结构域抗体、多特异性抗体(例如双特异性抗体、三特异性抗体或四特异性抗体)、dsFv(二硫键稳定化的Fv)、ScdsFv(二硫键稳定化的单链Fv抗体)。
在一些实施方案中，本公开的抗体-药物偶联物或其药学上可接受的盐或溶剂化物中的抗IL-4R的抗体的抗原结合片段是与上述的抗IL-4R的抗体的抗原结合片段结合相同的IL-4R或其表位。
在一些实施方案中，提供编码如上所述的抗IL-4R的抗体或其抗原结合片段的多核苷酸，例如DNA或RNA。
在一些实施方案中，提供包含上述多核苷酸的表达载体，例如真核表达载体、原核表达载体、病毒载体。
在一些实施方案中，提供上述表达载体转化的宿主细胞，例如真核细胞、原核细胞。一些具体实施方案中，宿主细胞为细菌(例如大肠杆菌)、酵母菌(例如毕赤酵母)、哺乳动物细胞(例如中国仓鼠卵巢(CHO)细胞或人胚肾(HEK)293细胞)。
在一些实施方案中，提供制备上述抗IL-4R的抗体或其抗原结合片段的方法包括步骤：在如前所述的宿主细胞中表达该抗体或其抗原结合片段，并自该宿主细胞中分离该抗体或其抗原结合片段。
本公开中抗IL-4R的抗体或其抗原结合片段中的氨基酸三字母代码和单字母代码如J.Biol.Chem，243，p3558(1968)中所述。
在某些实施方案中，k为1-10之间的任意数值，优选2-5之间的任意数值。k可以为整数，也可以为小数。
在某些实施方案中，连接子在细胞外是稳定的，使得ADC在存在于细胞外环境中时保持完整，但在细胞中内化时能够裂解。在一些实施方案中，当ADC进入表达对ADC的抗体部分具有特异性的抗原的细胞时，糖皮质激素药物部分从抗体部分裂解，且裂解释放糖皮质激素的未修饰形式。
在某些实施方案中，连接子中的可裂解部分为可裂解肽部分。在一些实施方案中，相对于包含其他可裂解部分的ADC，包含可裂解肽部分的ADC显示较低的聚集水平，改善的抗体与药物比率。在某些实施方案中，相对于不可裂解的连接子，添加可裂解部分增加细胞毒性和/或效力。在一些实施方案中，可裂解肽部分能够由酶裂解，且连接子为酶能够裂解的连接子。在一些实施方案中，酶为组织蛋白酶，且连接子为组织蛋白酶能够裂解的连接子。在某些实施方案中，与其它分裂机制相比，酶能够裂解的连接子(例如组织蛋白酶能够裂解的连接子)显示上述改善特性中的一种或多种。
在一些实施方案中，连接子包含拉伸单元，其一端通过碳原子与抗体共价连接而另一端与氨基酸单元、二硫化物部分、磺酰胺部分或非肽化学部分相连的化学结构片段。示例性拉伸单元包括但不限于
在一些实施方案中，连接子包含氨基酸单元，所述氨基酸单元优选包含由2至7个选自苯丙氨酸、甘氨酸、缬氨酸、赖氨酸、瓜氨酸、丝氨酸、谷氨酸、天冬氨酸、高赖氨酸、n-甲基-缬氨酸、(q为1-6的整数)的氨基酸构成的肽残基，示例性氨基酸单元包括但不限于缬氨酸-瓜氨酸(Val-Cit)、丙氨酸-苯丙氨酸(Ala-Phe)；苯丙氨酸-赖氨酸(Phe-Lys)、苯丙氨酸-高赖氨酸(Phe-Homolys)、n-甲基-缬氨酸-瓜氨酸(Me-Val-Cit)、丙氨酸-丙氨酸(Ala-Ala)、甘氨酸-谷氨酸(Gly-Glu)、谷氨酸-丙氨酸-丙氨酸(Glu-Ala-Ala)和甘氨酸-赖氨酸(Gly-Lys)、甘氨酸-缬氨酸-瓜氨酸(Glv-Val-Cit)和甘氨酸-甘氨酸-甘氨酸(Gly-Gly-Gly)、
在一些实施方案中，连接子可以包含至少一种聚乙二醇(PEG)部分。PEG部分可以例如包含-(PEG)p1-，其中p1为整数1至20，例如(PEG)2；(PEG)4；(PEG)5。
在一些实施方案中，所述连接子包含连接于D的间隔单元。在一些实施方案中，所述间隔单元包含对氨基苯甲氧基羰基(PAB)，
在一些实施方案中，所述间隔单元包含对氨基苯甲酰基，
在一些实施方案中，所述间隔单元包含：
-NH(CH2)n1-La-Lb-Lc-，其中n1选自0-6之间整数；La表示-C(＝O)-NH-、-NR7-(CH2)n2-、-O-或单键，R7选自氢、C1-6烷基、-(CH2)n3-COOH、-(CH2)n4-OH，n3选自1-4之间整数，n4选自1-6之间整数；Lb表示-CR8(R9)-、-O-、-NR10-或单键，R8和R9独立选自氢、C1-6烷基、C3-6环烷基、-(CH2)n5-NH2、-(CH2)n6-COOH、-(CH2)n7-OH，R10选自氢或C1-6烷基，n5选自0-6之间整数，n6选自1-4之间整数，n7选自1-4之间整数，且n5为0时，R8与R9不相同，或者R8和R9与其相连接的碳原子一起形成C3-6环烷基；Lc表示-CH2-或-C(＝O)-。
在一些实施方案中，-NH(CH2)n1-La-Lb-Lc-中Lc表示-NHCH2-。
在一些实施方案中，-NH(CH2)n1-La-Lb-Lc-中R8和R9独立选自氢、C1-6烷基、C3-6环烷基，例如氢、甲基、乙基或环丙基。
在一些实施方案中，-NH(CH2)n1-La-Lb-Lc-中La表示-O-或单键，Lb表示-CR8(R9)-或单键，Lc表示-C(＝O)-，R8和R9与其相连接的碳原子一起形成C3-6环烷基。
在一些实施方案中，-NH(CH2)n1-La-Lb-Lc-中La表示-O-或单键，Lb表示-CR8(R9)-或单键，Lc表示-C(＝O)-，R8和R9独立选自氢、C1-6烷基、C3-6环烷基、-(CH2)n5-NH2、-(CH2)n6-COOH、-(CH2)n7-OH，R10选自氢或C1-6烷基，n5选自0-6之间整数，n6选自1-4之间整数，n7选自1-4之间整数，且n5为0时，R8与R9不相同。
在一些实施方案中，-NH(CH2)n1-La-Lb-Lc-中La表示-O-或单键，Lb表示-CR8(R9)-或单键，Lc表示-CH2-。
在一些实施方案中，-NH(CH2)n1-La-Lb-Lc-中La表示-O-或单键，Lb表示-CR8(R9)-或单键，Lc表示-CH2-，R8和R9独立选自氢、C1-6烷基、C3-6环烷基，优选氢、甲基、乙基或环丙基。
在一些实施方案中，抗体-药物偶联物中-NH(CH2)n1-La-Lb-Lc-为：-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)-、
在一些实施方案中，-NH-(CH2)n1-La-Lb-Lc-为-NHCH2-或-NHCH2CH2CH2-。
在一些实施方案中，连接子中的间隔子单元包含(PEG)2。在一些实施方案中，尽管连接子长度较短，但相对于包含较长间隔子单元(例如(PEG)8)的ADC，包含较短间隔子单元(例如(PEG)2)的ADC显示较低的聚集水平和/或较高的药物负载。
另一方面，本公开抗体偶联物(ADC)中L-D是由下式表示的化学部分：
-Str-Pep-Sp-D
Str是与Ab共价连接的伸展基单元，
Sp为间隔单元，
Pep选自氨基酸单元、二硫化物部分、磺酰胺部分或以下非肽化学部分：
其中，W是-NH-亚杂环烷基-或杂环烷基；Y是亚杂芳基、亚芳基、-C(O)C1-6亚烷基、C2-6亚烯基、C1-6亚烷基或-C1-6亚烷基-NH-；每个R16独立选自C1-6烷基、C2-6烯基、-(C1-6亚烷基)NHC(NH)NH2或-(C1-6亚烷基)NHC(O)NH2；R17和R18各自独立选自氢、C1-6烷基、C2-6烯基、芳基、杂芳基，或R17和R18一起可形成C3-6环烷基；R19和R20各自独立选自C1-6烷基、C2-6烯基、芳基、杂芳基、(C1-6烷基)OCH2-，或R19和R20一起可形成C3-6环烷基环。
在一些实施方案中，所述抗体-药物偶联物(ADC)中Str选自下式表示的化学部分：
其中R21选自-W1-C(O)-、-C(O)-W1-C(O)-、-(CH2CH2O)p1C(O)-、-(CH2CH2O)p1CH2C(O)-、-(CH2CH2O)p1CH2CH2C(O)-，其中W1选自C1-6亚烷基、C1-6亚烷基-环烷基或1至8个原子的直链杂烷基，所述杂烷基包含1至3个选自N、O或S的杂原子，其中所述烷基、环烷基和直链杂烷基各自独立地任选被一个或多个选自卤素、氘、羟基、氰基、氨基、C1-6烷基、卤代C1-6烷基、氘代C1-6烷基、C1-6烷氧基和C3-6环烷基所取代，p1各自独立地为1至20的整数；
L1选自-NR22-(CH2CH2O)p2CH2CH2C(O)-、-NR22-(CH2CH2O)p2CH2C(O)-、-S(CH2)p2C(O)-、-(CH2)p2C(O)-或单键，优选单键；p2为1至20的整数，R22选自氢原子、C1-6烷基、卤代C1-6烷基或氘代C1-6烷基。
在一些实施方案中，所述抗体-药物偶联物Str中R21选自C1-6亚烷基C(O)-、-(CH2-CH2O)2C(O)-、-(CH2-CH2O)2CH2C(O)-、-(CH2-CH2O)2CH2CH2C(O)-、-(CH2-CH2O)3C(O)-和-(CH2-CH2O)4C(O)-。
在一些实施方案中，所述的Pep选自缬氨酸-瓜氨酸(Val-Cit)、丙氨酸-丙氨酸-天冬酰胺(Ala-Ala-Asn)、甘氨酸-甘氨酸-赖氨酸(Gly-Gly-lys)、缬氨酸-赖氨酸(Val-lys)、缬氨酸-丙氨酸(Val-Ala)、缬氨酸-苯丙氨酸(Val-Phe)或甘氨酸-甘氨酸-苯丙氨酸-甘氨酸(Gly-Gly-Phe-Gly)。
在一些实施方案中，所述的Sp选自-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)-、
在一些实施方案中，所述抗体-药物偶联物中连接子L包含：顺丁烯二酰亚胺-(PEG)4-CH2CH2C(O)-Gly-Gly-Phe-Gly、顺丁烯二酰亚胺-(PEG)2-Val-Cit、顺丁烯二酰亚胺-(PEG)6-Val-Cit、顺丁烯二酰亚胺-(PEG)8-Val-Cit、顺丁烯二酰亚胺-(PEG)4-CH2CH2C(O)-Val-lys、顺丁烯二酰亚胺-(CH2)5-Val-Cit、顺丁烯二酰亚胺-(CH2)5-Val-lys、顺丁烯二酰亚胺-(CH2)5-Gly-Gly-Phe-Gly、顺丁烯二酰亚胺-(PEG)4-CH2CH2C(O)-Gly-Gly-Phe-Gly、顺丁烯二酰亚胺-(PEG)2-Ala-Ala-Asn、顺丁烯二酰亚胺-(PEG)6-Ala-Ala-Asn、顺丁烯二酰亚胺-(PEG)8-Ala-Ala-Asn、顺丁烯二酰亚胺-(PEG)4-三唑-(PEG)3-磺酰胺、顺丁烯二酰亚胺-(PEG)2-CH2CH2C(O)-Val-lys、顺丁烯二酰亚胺-(PEG)4-三唑-(PEG)3-磺酰胺或Mal-(PEG)4-三唑-(PEG)3-二硫化物。
在另一些实施方案中，本公开抗体-药物偶联物(ADC)，其由下式表示：
其中k选自1至10，可以为整数，也可以为小数，p1选自2、4、6或8，p3、p4各自独立地选自0、1或2；
其中k选自1至10，可以为整数，也可以为小数，p1选自2、4、6或8，p3、p4各自独立地选自0、1或2；
在另一些实施方式中，本公开抗体-药物偶联物(ADC)，选自：

其中k选自1至10，可以为整数，也可以为小数。
本公开还提供了一种式II-A所示化合物或其可药用盐，
其中p1选自2、4、6或8，p3、p4各自独立地选自0、1或2。本公开还提供了一种式II-B所示化合物或其可药用盐，
其中X为卤素，p1选自2、4、6或8，p3、p4各自独立地选自0、1或2。
本公开所述的结构中，表示单键或双键。
本公开还提供了一种药物组合物，包括至少一种前述抗体-药物偶联物，以及药学上可接受的载体、稀释剂或赋形剂。
在一些实施方案中，所述的药物组合物的单位剂量为0.001mg-1000mg。
在一些实施方案中，基于组合物的总重量，所述的药物组合物含有0.01％-99.99％的前述化合物。在一些实施方案中，所述的药物组合物含有0.1％-99.9％的前述化合物。在一些实施方案中，所述的药物组合物含有0.5％-99.5％的前述化合物。在一些实施方案中，所述的药物组合物含有1％-99％的前述化合物。在一些实施方案中，所述的药物组合物含有2％-98％的前述化合物。
在一些实施方案中，基于组合物的总重量，所述的药物组合物含有0.01％-99.99％的药学上可接受的载体、稀释剂或赋形剂。在一些实施方案中，所述的药物组合物含有0.1％-99.9％的药学上可接受的载体、稀释剂或赋形剂。在一些实施方案中，所述的药物组合物含有0.5％-99.5％的药学上可接受的载体、稀释剂或赋形剂。在一些实施方案中，所述的药物组合物含有1％-99％的药学上可接受的载体、稀释剂或赋形剂。在一些实施方案中，所述的药物组合物含有2％-98％的药学上可接受的载体、稀释剂或赋形剂。
本公开的抗体-药物偶联物可以用适合适应症的方式对患者施用，例如，肠胃外、局部或通过吸入施用。如果注射的话，拮抗剂可以通过例如，关节内、静脉内、肌内、损伤内、腹膜内或皮下路径，通过快速浓注或连续输注进行施用。在疾病或损伤处的局部给药被认为是经皮递送和从植入物中持续释放。通过吸入递送包括，例如，鼻或口腔吸入、使用喷雾器、吸入气雾剂形式的拮抗剂等。其它的选择包括眼药水；口服制剂，包括丸剂、糖浆剂、锭剂或口香糖；和局部制剂如洗剂、凝胶剂、喷雾剂和软膏剂。
本公开还提供了一种可吸入的药物组合物，包括式(I)所示的抗体-药物偶联物以及药学上可接受的载体，
Ab-(L-D)k
(I)
其中，Ab为抗IL-4R的抗体或其抗原结合片段，
L为将Ab共价连接于D的连接子，且k为1至20(包括1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或任意两数值之间任意数值)，
D为糖皮质激素或其残基。
可选的Ab、L、D如前文所述。
在一些实施方案中，Ab为抗IL-4R的Fab片段。
本公开还提供了所述的抗体-药物偶联物和/或包含抗体-药物偶联物的药物组合物在制备用于治疗或预防IL-4R介导的疾病或病症的药物中的用途。
本公开还提供了所述的抗体-药物偶联物和/或包含抗体-药物偶联物的药物组合物在制备用于治疗或预防免疫性疾病或病症的药物中的用途。所述疾病或病症选自：哮喘、鼻息肉、慢性鼻窦炎、过敏性皮肤病、嗜酸细胞性食管炎、慢性阻塞性肺病、过敏性鼻炎、关节炎、炎症性疾病、变应性反应、自体免疫淋巴组织增生性综合征、自体免疫性溶血性贫血、巴雷特食管、自体免疫葡萄膜炎、结核病和肾病。优选哮喘或过敏性皮肤病。
本公开还提供了一种向表达IL-4R的细胞递送糖皮质激素的方法，包括使表达IL-4R的细胞与本公开所述的抗体-药物偶联物接触的步骤。
本公开进一步提供了一种试剂盒，其包含本公开所述的抗体-药物偶联物，或药物组合物。
发明的详细说明
除非另有限定，本公开所用的所有技术和科学术语均与本公开所属领域普通技术人员的通常理解一致。虽然也可采用与本公开所述相似或等同的任何方法和材料实施或测试本公开，但本公开描述了优选的方法和材料。描述和要求保护本公开时，依据以下定义使用下列术语。
当本公开中使用商品名时，申请人旨在包括该商品名产品的制剂、该商品名产品的非专利药和活性药物部分。
除非有相反陈述，在说明书和权利要求书中使用的术语具有下述含义。
术语“连接子”、“连接单元”、“接头单元”、“接头”或“连接片段”是指一端与配体连接而另一端与药物相连的化学结构片段或键，也可以连接其他接头后再与药物相连。
接头可以包含一种或多种接头构件。例示性的接头构件包括6-马来酰亚氨基己酰基(MC)、马来酰亚氨基丙酰基(MP)、缬氨酸-瓜氨酸(Val-Cit或vc)、丙氨酸-苯丙氨酸(ala-phe)、对氨基苄氧羰基(PAB)，及那些源自与接头试剂的偶联的：N-琥珀酰亚氨基4-(2-吡啶基硫代)戊酸酯(SPP)、N-琥珀酰亚氨基4-(N-马来酰亚氨基甲基)环己烷-1羧酸酯(SMCC，在本文中也称作MCC)和N-琥珀酰亚氨基(4-碘-乙酰基)氨基苯甲酸酯(SIAB)。接头可以包括拉伸单元、间隔单元、氨基酸单元和延伸单元。可以通过本领域已知方法合成，诸如US2005-0238649A1中所记载的。接头可以是便于在细胞中释放药物的“可切割接头”。例如，可使用酸不稳定接头(例如腙)、蛋白酶敏感(例如肽酶敏感)接头、光不稳定接头、二甲基接头、或含二硫化物接头(Chari等,Cancer Research 52:127-131(1992)；美国专利No.5,208,020)。
术语“拉伸单元”指一端通过碳原子与抗体共价连接而另一端与氨基酸单元、二硫化物部分、磺酰胺部分或非肽化学部分相连的化学结构片段。
术语“间隔单元”是一种双功能化合结构片段，可用于偶联氨基酸单元和糖皮质激素最终形成抗体-药物偶联物，这种偶联方式可以将糖皮质激素选择性的连接到氨基酸单元上。
术语“氨基酸”是指分子结构中含有氨基和羧基，并且氨基和羧基都直接连接在-CH-结构上的有机化合物。通式是H2NCHRCOOH，R为H、取代或未取代烷基等。根据氨基连结 在羧酸中碳原子的位置，可分为α、β、γ、δ、ε……-氨基酸。在生物界中，构成天然蛋白质的氨基酸具有其特定的结构特点，即其氨基直接连接在α-碳原子上，即α-氨基酸，包括甘氨酸(Glycine)、丙氨酸(Alanine)、缬氨酸(Valine)、亮氨酸(Leucine)、异亮氨酸(Isoleucine)、苯丙氨酸(Phenylalanine)、色氨酸(Tryptophan)、酪氨酸(Tyrosine)、天冬氨酸(Aspartic acid)、组氨酸(Histidine)、天冬酰胺(Asparagine)、谷氨酸(Glutamic acid)、赖氨酸(Lysine)、谷氨酰胺(Glutamine)、甲硫氨酸(Methionine)、精氨酸(Arginine)、丝氨酸(Serine)、苏氨酸(Threonine)、半胱氨酸(Cysteine)、脯氨酸(Proline)等。非天然氨基酸如瓜氨酸。如本领域技术人员所公知的，非天然氨基酸并不构成天然蛋白质，因此也不参与本公开中抗体的合成。本公开所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中所述。

术语“抗体-药物偶联物”，指配体通过稳定的连接单元与具有生物活性的药物相连。在本公开中“抗体-药物偶联物”(antibody drug conjugate，ADC)，指把单克隆抗体或者抗体片段通过稳定的连接单元与具有生物活性的糖皮质激素相连。其中抗体或抗体片段可通过其中的特定基团(例如链间二硫键)与包含接头的糖皮质激素分子相结合。
术语“载药量”是指抗体-药物偶联物群体中，每个抗体-药物偶联物分子载有的药物平均数量，也可以表示为药物量和抗体量的比值。载药量的范围可以是每个抗体(Ab)连接1-20个，优选1-10个糖皮质激素(D)。在本公开的实施方式中，载药量表示为k，示例性的可以为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或任意两数值之间数值的均值。优选1-10，更优选1-8，或2-8，或2-7，或3-8，或3-7，或3-6，或4-7，或4-6，或4-5的均值。可用常规方法如UV/可见光光谱法、质谱、ELISA试验、单抗分子大小变异体测定法(CE-SDS)和HPLC特征鉴定偶联反应后每个ADC分子的药物平均数量。
本公开单抗分子大小变异体测定法(CE-SDS)可采用十二烷基硫酸钠毛细管电泳(CE-SDS)紫外检测方法，在还原和非还原条件下，依据分子量大小，按毛电泳法(2015年版《中国药典》0542)，定量测定重组单克隆抗体产品的纯度。
本公开的一个实施方式中，糖皮质激素通过连接单元偶联在配体的N端氨基和/或赖氨酸残基的ε-氨基上，一般地，偶联反应中能与抗体偶联的药物分子数将小于理论上的最大值。
可以用以下非限制性方法控制抗体-药物偶联物的载量，包括：
(1)控制连接试剂和单抗的摩尔比，
(2)控制反应时间和温度，
(3)选择不同的反应试剂。
术语“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链、和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(Fv区)；靠近C端的其余氨基酸序列相对稳定，为恒定区。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(LCVR)和重链可变区(HCVR)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1、LCDR2、和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。
本公开的抗体包括鼠源抗体、嵌合抗体、人源化抗体和全人源抗体，优选人源化抗体和全人源抗体。
术语“鼠源抗体”在本公开中为根据本领域知识和技能用鼠制备抗体。制备时用特定抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。
术语“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要先建立分泌鼠源性特异性单抗的杂交瘤，然后从鼠杂交瘤细胞中克隆可变区基因，再根据需要克隆人抗体的恒定区基因，将鼠可变区基因与人恒定区基因连接成嵌合基因后插入表达载体中，最后在真核系统或原核系统中表达嵌合抗体分子。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体框架序列中产生的抗体。可以克服嵌合抗体由于携带大量鼠蛋白成分，从而诱导的异源性反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库(在因特网www.mrccpe.com.ac.uk/vbase可获得)，以及在Kabat，E.A.等人，1991 Sequences of Proteins of Immunological Interest，第5版中找到。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。本公开的人源化抗体也包括进一步由噬菌体展示对CDR进行亲和力成熟后的人源化抗体。进一步描述参与人源化可使用小鼠抗体的方法的文献包括，例如Queen等，Proc.，Natl.Acad.Sci.USA，88，2869，1991和Winter及其同事的方法[Jones等，Nature，321，522(1986)，Riechmann，等，Nature， 332，323-327(1988)，Verhoeyen，等，Science，239，1534(1988)]。
术语“全人源抗体”、“全人抗体”或“完全人源抗体”，也称“全人源单克隆抗体”，其抗体的可变区和恒定区都是人源的，去除免疫原性和毒副作用。单克隆抗体的发展经历了四个阶段，分别为：鼠源性单克隆抗体、嵌合性单克隆抗体、人源化单克隆抗体和全人源单克隆抗体。本公开为全人源单克隆抗体。全人抗体制备的相关技术主要有：人杂交瘤技术、EBV转化B淋巴细胞技术、噬菌体显示技术(phage display)、转基因小鼠抗体制备技术(transgenic mouse)和单个B细胞抗体制备技术等。
术语“抗原结合片段”是指抗体的保持特异性结合抗原的能力的一个或多个片段。已显示可利用全长抗体的片段来进行抗体的抗原结合功能。“抗原结合片段”中包含的结合片段的实例包括(i)Fab片段，由VL、VH、CL和CH1结构域组成的单价片段；(ii)F(ab')2片段，包含通过铰链区上的二硫桥连接的两个Fab片段的二价片段；(iii)由VH和CH1结构域组成的Fd片段；(iv)由抗体的单臂的VH和VL结构域组成的Fv片段；(v)单结构域或dAb片段(Ward等人,(1989)Nature341：544-546)，其由VH结构域组成；和(vi)分离的互补决定区(CDR)或(vii)可任选地通过合成的接头连接的两个或更多个分离的CDR的组合。此外，虽然Fv片段的两个结构域VL和VH由分开的基因编码，但可使用重组方法，通过合成的接头连接它们，从而使得其能够产生为其中VL和VH区配对形成单价分子的单个蛋白质链(称为单链Fv(scFv)；参见，例如，Bird等人(1988)Science242:423-426；和Huston等人(1988)Proc.Natl.Acad.Sci USA85:5879-5883)。此类单链抗体也意欲包括在术语抗体的“抗原结合片段”中。使用本领域技术人员已知的常规技术获得此类抗体片段，并且以与对于完整抗体的方式相同的方式就功用性筛选片段。可通过重组DNA技术或通过酶促或化学断裂完整免疫球蛋白来产生抗原结合部分。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1、IgG2、IgG3或IgG4亚型)、IgA1、IgA2、IgD、IgE或IgM抗体。
Fab是通过用蛋白酶木瓜蛋白酶(切割H链的224位的氨基酸残基)处理IgG抗体分子所获得的片段中的具有约50,000的分子量并具有抗原结合活性的抗体片段，其中H链N端侧的约一半和整个L链通过二硫键结合在一起。
F(ab')2是通过用酶胃蛋白酶消化IgG铰链区中两个二硫键的下方部分而获得的分子量为约100,000并具有抗原结合活性并包含在铰链位置相连的两个Fab区的抗体片段。
Fab'是通过切割上述F(ab')2的铰链区的二硫键而获得的分子量为约50,000并具有抗原结合活性的抗体片段。
此外，可以通过将编码抗体的Fab'片段的DNA插入到原核生物表达载体或真核生物表达载体中并将载体导入到原核生物或真核生物中以表达Fab'来生产所述Fab'。
术语“单链抗体”、“单链Fv”或“scFv”意指包含通过接头连接的抗体重链可变结构域(或区域；VH)和抗体轻链可变结构域(或区域；VL)的分子。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成，例如使用1-4个重复的变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA90:6444-6448)。可用于本公开的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immuno l.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061， Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。
术语“CDR”是指抗体的可变结构域内主要促成抗原结合的6个高变区之一。所述6个CDR的最常用的定义之一由Kabat E.A.等人，(1991)Sequences of proteins of immunological interest.NIH Publication91-3242)提供。如本文中使用的，CDR的Kabat定义只应用于轻链可变结构域的CDR1、CDR2和CDR3(CDR L1、CDR L2、CDR L3或L1、L2、L3)，以及重链可变结构域的CDR2和CDR3(CDR H2、CDR H3或H2、H3)。通常，每个重链可变区中存在三个CDR(HCDR1、HCDR2、HCDR3)，每个轻链可变区中存在三个CDR(LCDR1、LCDR2、LCDR3)。可以使用各种公知方案中的任何一种来确定CDR的氨基酸序列边界，包括“Kabat”编号规则(参见Kabat等(1991)，“Sequences of Proteins of Immunological Interest”，第5版，Public Health Service，National Institutes of Health，Bethesda，MD)、“Chothia”编号规则(参见Al-Lazikani等人，(1997)JMB 273：927-948)和ImMunoGenTics(IMGT)编号规则(参见Lefranc M.P.，Immunologist，7，132-136(1999)；Lefranc，M.P.等，Dev.Comp.Immunol.，27，55-77(2003))等。例如，对于经典格式，遵循Kabat规则，所述重链可变域(VH)中的CDR氨基酸残基编号为31-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)；轻链可变域(VL)中的CDR氨基酸残基编号为24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)。遵循Chothia规则，VH中的CDR氨基酸编号为26-32(HCDR1)、52-56(HCDR2)和95-102(HCDR3)；并且VL中的氨基酸残基编号为26-32(LCDR1)、50-52(LCDR2)和91-96(LCDR3)。通过组合Kabat和Chothia两者的CDR定义，CDR由人VH中的氨基酸残基26-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)和人VL中的氨基酸残基24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)构成。遵循IMGT规则，VH中的CDR氨基酸残基编号大致为26-35(CDR1)、51-57(CDR2)和93-102(CDR3)，VL中的CDR氨基酸残基编号大致为27-32(CDR1)、50-52(CDR2)和89-97(CDR3)。遵循IMGT规则，抗体的CDR区可以使用程序IMGT/DomainGap Align确定。
术语“抗体框架”，是指可变结构域VL或VH的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。从本质上讲，其是不具有CDR的可变结构域。
“与IL-4R结合”，指能与人IL-4R(或其表位、片段)相互作用。本文的术语“抗原结合位点”指由本文抗体或抗原结合片段识别的三维空间位点。
术语“表位”或“抗原决定簇”是指抗原上免疫球蛋白或抗体特异性结合的部位。表位通常以独特的空间构象包括至少3、4、5、6、7、8、9、10、11、12、13、14或15个连续或非连续的氨基酸(参见，例如，Epitope Mapping Protocols in Methods in Molecular B iology，第66卷，G.E.Morris，Ed.(1996))。
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体对预先确定的抗原上的表位的结合。通常，抗体以大约小于10-7M，例如：大约小于10-8M、10-9M或10-10M或更小的亲和力(KD)结合。
术语“核酸分子”是指DNA分子和RNA分子。核酸分子可以是单链或双链的，但优选是双链DNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。
术语“载体”是指能够运输已与其连接的另一个核酸的核酸分子。在一个实施方案中，载体 是“质粒”，其是指可将另外的DNA区段连接至其中的环状双链DNA环。在另一个实施方案中，载体是病毒载体，其中可将另外的DNA区段连接至病毒基因组中。本文中公开的载体能够在已引入它们的宿主细胞中自主复制(例如，具有细菌的复制起点的细菌载体和附加型哺乳动物载体)或可在引入宿主细胞后整合入宿主细胞的基因组，从而随宿主基因组一起复制(例如，非附加型哺乳动物载体)。
现有技术中熟知生产和纯化抗体和抗原结合片段的方法，如冷泉港的抗体实验技术指南，5-8章和15章。抗原结合片段同样可以用常规方法制备。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人源FR区。人FR种系序列可以通过比对IMGT人类抗体可变区种系基因数据库和MOE软件，从ImMunoGeneTics(IMGT)的网站http://imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可包括细菌、微生物、植物或动物细胞。易于转化的细菌包括肠杆菌科(enterobacteriaceae)的成员，例如大肠杆菌(Escherichia coli)或沙门氏菌(Salmonella)的菌株；芽孢杆菌科(Bacillaceae)例如枯草芽孢杆菌(Bacillus subtilis)；肺炎球菌(Pneumococcus)；链球菌(Streptococcus)和流感嗜血菌(Haemophilus influenzae)。适当的微生物包括酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)。适当的动物宿主细胞系包括CHO(中国仓鼠卵巢细胞系)和NS0细胞。
本公开工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端位点。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化。比如，用含调整过的缓冲液的A或GSepharose FF柱进行纯化。洗去非特异性结合的组分。再用PH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
氨基酸序列“同一性”指在比对氨基酸序列及必要时引入间隙，以达成最大序列同一性百分比，且不将任何保守性取代视为序列同一性的一部分，第一序列中与第二序列中的氨基酸残基同一的氨基酸残基的百分比。为测定氨基酸序列同一性百分比的目的，比对可以通过属于本领域技术的范围内的多种方式来实现，例如使用公开可得到的计算机软件，诸如BLAST、BLAST-2、ALIGN、ALIGN-2或Megalign(DNASTAR)软件。本领域技术人员可确定适用于测量比对的参数，包括在所比较的序列全长上达成最大比对所需的任何算法。
术语“抗IL-4R抗体”或“与IL-4R结合的抗体”是指能够例如以足够的亲和力结合IL-4R的抗体，使得该抗体可用作靶向IL-4R的治疗剂。抗IL-4R抗体与不相关的非IL-4R蛋白的结合程度可以小于如，例如，通过放射免疫测定(RIA)所测量的抗体与IL-4R的结合的约10％。在一些实施方案中，与IL-4R结合的抗体具有≤1μM、≤100nM、≤10nM、≤1nM或≤0.1nM的解离常数(Kd)。
术语“肽”是指介于氨基酸和蛋白质之间的化合物片段，由2个或2个以上氨基酸分子通过 肽键相互连接而成，是蛋白质的结构与功能片段，如激素、酶类等本质上都是肽。
术语“糖”是指由C、H、O三种元素组成的生物大分子，可分为单糖、二糖和多糖等。
术语“荧光探针”是指在紫外-可见-近红外区有特征荧光，并且其荧光性质(激发和发射波长、强度、寿命和偏振等)可随所处环境的性质，如极性、折射率、粘度等改变而灵敏地改变的一类荧光性分子，其与核酸(DNA或RNA)、蛋白质或其他大分子结构非共价相互作用而使一种或几种荧光性质发生改变，可用于研究大分子物质的性质和行为。
术语“烷基”指饱和脂肪族烃基团，其为包含1至20个碳原子的直链或支链基团，优选含有1至12个碳原子的烷基。非限制性实例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基、正庚基、2-甲基己基、3-甲基己基、4-甲基己基、5-甲基己基、2,3-二甲基戊基、2,4-二甲基戊基、2,2-二甲基戊基、3,3-二甲基戊基、2-乙基戊基、3-乙基戊基、正辛基、2,3-二甲基己基、2,4-二甲基己基、2,5-二甲基己基、2,2-二甲基己基、3,3-二甲基己基、4,4-二甲基己基、2-乙基己基、3-乙基己基、4-乙基己基、2-甲基-2-乙基戊基、2-甲基-3-乙基戊基、正壬基、2-甲基-2-乙基己基、2-甲基-3-乙基己基、2,2-二乙基戊基、正癸基、3,3-二乙基己基、2,2-二乙基己基，及其各种支链异构体等。更优选的是含有1至6个碳原子的烷基，非限制性实施例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基等。烷基可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氧代基、羧基或羧酸酯基。
术语“杂烷基”指含有一个或多个选自N、O或S的杂原子的烷基，其中烷基如上所定义。
术语“环烷基”指饱和或部分不饱和单环或多环环状烃取代基，环烷基环包含3至20个碳原子，优选包含3至12个碳原子，更优选包含3至6个碳原子。单环环烷基的非限制性实例包括环丙基、环丁基、环戊基、环戊烯基、环己基、环己烯基、环己二烯基、环庚基、环庚三烯基、环辛基等；多环环烷基包括螺环、稠环和桥环的环烷基。“碳环”指的是环烷基中的环系。
术语“螺环烷基”指5至20元的单环之间共用一个碳原子(称螺原子)的多环基团，其可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统。优选为6至14元，更优选为7至10元。根据环与环之间共用螺原子的数目将螺环烷基分为单螺环烷基、双螺环烷基或多螺环烷基，优选为单螺环烷基和双螺环烷基。更优选为4元/4元、4元/5元、4元/6元、5元/5元或5元/6元单螺环烷基。“螺碳环”指的是螺环烷基中的环系。螺环烷基的非限制性实 例包括：
术语“稠环烷基”指5至20元，系统中的每个环与体系中的其他环共享毗邻的一对碳原子的全碳多环基团，其中一个或多个环可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统。优选为6至14元，更优选为7至10元。根据组成环的数目可以分为双环、三环、四环或多环稠环烷基，优选为双环或三环，更优选为5元/5元或5元/6元双环烷基。“稠碳环”指的是稠环烷基中的环系。稠环烷基的非限制性实例包括：
术语“桥环烷基”指5至20元，任意两个环共用两个不直接连接的碳原子的全碳多环基团，其可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统。优选为6至14元，更优选为7至10元。根据组成环的数目可以分为双环、三环、四环或多环桥环烷基，优选为双环、三环或四环，更有选为双环或三环。桥环烷基的非限制性实例包括：
所述环烷基环可以稠合于芳基、杂芳基或杂环烷基环上，其中与母体结构连接在一起的环为环烷基，非限制性实例包括茚满基、四氢萘基、苯并环庚烷基等。环烷基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氧代基、羧基或羧酸酯基。
术语“杂环基”指饱和或部分不饱和单环或多环环状烃取代基，其包含3至20个环原子，其中一个或多个环原子为选自氮、氧或S(O)m(其中m是整数0至2)的杂原子，但不包括-O-O-、-O-S-或-S-S-的环部分，其余环原子为碳。优选包含3至12个环原子，其中1～4个是杂原子；更优选包含3至6个环原子。单环杂环基的非限制性实例包括吡咯烷基、咪唑烷基、四氢呋喃基、四氢噻吩基、二氢咪唑基、二氢呋喃基、二氢吡唑基、二氢吡咯基、哌啶基、哌嗪基、吗啉基、硫代吗啉基、高哌嗪基等，优选哌啶基、吡咯烷基。多环杂环基包括螺环、稠环和桥环的杂环基。“杂环”指的是杂环基中的环系。
术语“螺杂环基”指5至20元的单环之间共用一个原子(称螺原子)的多环杂环基团，其中一个或多个环原子为选自氮、氧或S(O)m(其中m是整数0至2)的杂原子，其余环原子为碳。其可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统。优选为6至14元，更优选为7至10元。根据环与环之间共用螺原子的数目将螺杂环基分为单螺杂环基、双螺杂环基或多螺杂环基，优选为单螺杂环基和双螺杂环基。更优选为4元/4元、4元/5元、4元/6元、5元/5元或5元/6元单螺杂环基。“螺杂环”指的是螺杂环基中的环系。螺杂环基的非限制性实例包括：
术语“稠杂环基”指5至20元，系统中的每个环与体系中的其他环共享毗邻的一对原子的多环杂环基团，一个或多个环可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统，其中一个或多个环原子为选自氮、氧或S(O)m(其中m是整数0至2)的杂原子，其余环原子为碳。优选为6至14元，更优选为7至10元。根据组成环的数目可以分为双环、三环、四环或多环稠杂环基，优选为双环或三环，更优选为5元/5元或5元/6元双环稠杂环基。“稠杂环”指的是稠杂环基中的环系。稠杂环基的非限制性实例包括：
术语“桥杂环基”指5至14元，任意两个环共用两个不直接连接的原子的多环杂环基团，其可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统，其中一个或多个环原子为选自氮、氧或S(O)m(其中m是整数0至2)的杂原子，其余环原子为碳。优选为6至14元，更优选为7至10元。根据组成环的数目可以分为双环、三环、四环或多环桥杂环基，优选为双环、三环或四环，更有选为双环或三环。桥杂环基的非限制性实例包括：
所述杂环基环可以稠合于芳基、杂芳基或环烷基环上，其中与母体结构连接在一起的环 为杂环基，其非限制性实例包括：
等。
杂环基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氧代基、羧基或羧酸酯基。
术语“芳基”指具有共轭的π电子体系的6至14元全碳单环或稠合多环(也就是共享毗邻碳原子对的环)基团，优选为6至10元，例如苯基和萘基。所述芳基环可以稠合于杂芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为芳基环。“芳环”指的是芳基中的环系。芳基非限制性实例包括：
芳基可以是取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、羧基或羧酸酯基，优选苯基。
术语“稠环芳基”可以是含有8-14个环原子由两个或两个以上环状结构彼此共用两个相邻的原子连接起来形成的不饱和的具有芳香性的稠环结构，环原子优选8-12个。例如包括全部不饱和稠环芳基，例如萘、菲等，还包括部分饱和稠环芳基，例如苯并3-8元饱和单环环烷基、苯并3-8元部分饱和单环环烷基。“稠芳环”指的是稠环芳基中的环系。稠环芳基具体实例如2,3-二氢-1H-茚基、IH-茚基、1,2,3,4-四氢萘基、1,4-二氢萘基等。
术语“杂芳基”指包含1至4个杂原子、5至14个环原子的杂芳族体系，其中杂原子选自氧、硫和氮。杂芳基优选为5至12元，例如咪唑基、呋喃基、噻吩基、噻唑基、吡唑基、噁唑基、吡咯基、四唑基、吡啶基、嘧啶基、噻二唑、吡嗪基等，优选为咪唑基、吡唑基、嘧啶基或噻唑基；更优选为吡唑基或噻唑基。所述杂芳基环可以稠合于芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为杂芳基环。“杂芳环”指的是杂芳基中的环系。杂芳基非限制性实例包括：

杂芳基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、羧基或羧酸酯基。
术语“稠杂芳基”可以是含有5-14个环原子(其中至少含有一个杂原子)由两个或两个以上环状结构彼此共用两个相邻的原子连接起来形成的不饱和的具有芳香性的稠环结构，同时包括碳原子、氮原子和硫原子可以被氧代，优选"5-12元稠杂芳基"、"7-12元稠杂芳基"、"9-12元稠杂芳基"等，例如苯并呋喃基、苯并异呋喃基、苯并噻吩基、吲哚基、异吲哚、苯并噁唑基、苯并咪唑基、吲唑基、苯并三唑基、喹啉基、2-喹啉酮、4-喹啉酮、1-异喹啉酮、异喹啉基、吖啶基、菲啶基、苯并哒嗪基、酞嗪基、喹唑啉基、喹喔啉基、酚嗪基、喋啶基、嘌呤基、萘啶基、吩嗪、吩噻嗪等。“稠杂芳环”指的是稠杂芳基中的环系。
稠杂芳基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、羧基或羧酸酯基。
术语“亚烷基(-CH2-)”则表示烷烃分子中去除2个氢原子后余下的部分，包括1至20个碳原子的直链和支链亚基团。含有1至6个碳原子的亚烷基，非限制性实施例包括亚甲基(-CH2-)、亚乙基(如-CH2CH2-或-CH(CH3)-)、亚丙基(如-CH2CH2CH2-或-CH(CH2CH3)-)、亚丁基(如-CH2CH2CH2CH2-)。亚烷基可以是取代的或未取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，优选一个或多个以下基团，独立地选自卤素、氘、羟基、硝基、氰基或氨基。同理，“亚烯基”、“亚杂芳基”、“亚芳基”、“亚杂环烷基”等如前所定义。
术语“亚链烯基”指包含具有2至8个碳原子，优选地具有2至6个碳原子，更优选地具有2至4个碳原子并在任何位置具有至少一个双键的线性链烯基，包括例如亚乙烯基、亚烯丙基(allylene)、亚丙烯基、亚丁烯基、亚异戊二烯基(prenylene)、亚丁二烯基(butadienylene)、亚戊烯基、亚戊二烯基、亚己烯基、亚己二烯基等。
术语“亚链炔基”包括具有2至8个碳原子，优选地具有2至6个碳原子，更优选地具有2至4个碳原子且在任何位置具有至少一个叁键的线性亚链炔基，包括例如亚乙炔基、亚丙炔基、亚丁炔基、亚戊炔基、亚己炔基等。
术语“烷氧基”指-O-(烷基)和-O-(非取代的环烷基)，其中烷基的定义如上所述。烷氧基的非限制性实例包括：甲氧基、乙氧基、丙氧基、丁氧基、环丙氧基、环丁氧基、环戊氧基、环己氧基。烷氧基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环 烷硫基、羧基或羧酸酯基。
术语“烷硫基”指-S-(烷基)和-S-(非取代的环烷基)，其中烷基的定义如上所述。烷硫基的非限制性实例包括：甲硫基、乙硫基、丙硫基、丁硫基、环丙硫基、环丁硫基、环戊硫基、环己硫基。烷硫基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基中的一个或多个取代基所取代。
术语“羟烷基”指被羟基取代的烷基，其中烷基如上所定义。
术语“卤代烷基”指被卤素取代的烷基，其中烷基如上所定义。
术语“氘代烷基”指被氘原子取代的烷基，其中烷基如上所定义。
术语“羟基”指-OH基团。
术语“氧代”指＝O基团。例如，碳原子与氧原子通过双键连接，其中形成酮或醛基。
术语“硫代”指＝S基团。例如，碳原子与硫原子通过双键连接，形成硫代羰基-C(S)-。
术语“卤素”指氟、氯、溴或碘。
术语“氨基”指-NH2。
术语“氰基”指-CN。
术语“硝基”指-NO2。
术语“羧基”指-C(O)OH。
术语“醛基”指-CHO。
术语“羧酸酯基”指-C(O)O(烷基)或-C(O)O(环烷基)，其中烷基、环烷基如上所定义。
术语“酰卤”指含有-C(O)-卤素的基团的化合物。
术语“磺酰基”指-S(O)(O)-。
术语“亚磺酰基”指-S(O)-。
“氨基保护基”是本领域已知的适当的用于氨基保护的基团，参见文献(“Protective Groups in Organic Synthesis”，5Th.Ed.T.W.Greene&P.G.M.Wuts)中的氨基保护基团，优选地，所述的氨基保护基可以是(C1-10烷基或芳香基)酰基，例如：甲酰基，乙酰基，苯甲酰基等；可以是(C1-6烷基或C6-10芳基)磺酰基；也可以是(C1-6烷氧基或C6-10芳基氧基)羰基，例如：Boc或Cbz；还可以是取代或非取代的烷基，例如：三苯甲基(Tr)、2,4-二甲氧基苄基(DMB)、对甲氧基苄基(PMB)或苄基(Bn)。
“任选”或“任选地”意味着随后所描述的事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。例如，“任选被烷基取代的杂环烷基团”意味着烷基可以但不必须存在，该说明包括杂环烷基团被烷基取代的情形和杂环烷基团不被烷基取代的情形。
术语“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，以及其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
术语“药物载体”用于本公开的药物，是指能改变药物进入人体的方式和在体内的分布、控制药物的释放速度并将药物输送到靶向器官的体系。药物载体释放和靶向系统能够减少药物 降解及损失，降低副作用，提高生物利用度。如可作为载体的高分子表面活性剂由于其独特的两亲性结构，可以进行自组装，形成各种形式的聚集体，优选的实例如胶束、微乳液、凝胶、液晶、囊泡等。这些聚集体具有包载药物分子的能力，同时又对膜有良好的渗透性，可以作为优良的药物载体。
术语“赋形剂”是在药物制剂中除主药以外的附加物，也可称为辅料。如片剂中的粘合剂、填充剂、崩解剂、润滑剂；半固体制剂软膏剂、霜剂中的基质部分；液体制剂中的防腐剂、抗氧剂、矫味剂、芳香剂、助溶剂、乳化剂、增溶剂、渗透压调节剂、着色剂等均可称为赋形剂。
术语“稀释剂”又称填充剂，其主要用途是增加片剂的重量和体积。稀释剂的加入不仅保证一定的体积大小，而且减少主要成分的剂量偏差，改善药物的压缩成型性等。当片剂的药物含有油性组分时，需加入吸收剂吸收油性物，使保持“干燥”状态，以利于制成片剂。
本公开中化合物可以含有一个或多个不对称中心，因此可以产生对映异构体、非对映异构体，并且其可以根据绝对立体化学定义为(R)-或(S)-或用于氨基酸的(D)-或(L)-的其它立体异构形式。本公开包括所有可能异构体以及其外消旋和光学纯的形式。光学活性的(+)和(-)、(R)-和(S)-或(D)-和(L)-异构体可以使用手性合成子或手性试剂制备，或者可以使用常规方法例如色谱法和分级结晶制备。用于制备/分离各个对映体的常规方法包括从合适的的光学纯前体手性合成或使用例如手性高效液相色谱法(HPLC)的外消旋物(或盐或衍生物的外消旋物)拆分。当本文描述的化合物含有烯双键或其它几何不对称性中心，除非另有说明，否则其意味着所述化合物包括E和Z几何异构体。而且，所有的互变异构形式也意味着包括在内。
本公开所述化合物的化学结构中，键表示未指定构型，即如果化学结构中存在手性异构体，键可以为或或者同时包含和两种构型。本公开所述化合物的化学结构中，键并未指定构型，即可以为Z构型或E构型，或者同时包含两种构型。
“立体异构体”指通过相同的键键合但具有不同的三维结构的相同原子组成的化合物，其不可互换。本公开中预期各种立体异构体及其混合物，并且包括“对映异构体”，其指其分子彼此为不能重叠的镜像的两种立体异构体。
“互变异构体”指质子从分子的一个原子转移到同一分子的另一个原子。本公开中包括任何所述化合物的互变异构体。
本公开所述化合物或其可药用盐、或其异构体的任何同位素标记的衍生物都被本公开所覆盖。能够被同位素标记的原子包括但不限于氢、碳、氮、氧、磷、氟、氯、碘等。它们可分别被同位素同位素2H(D)、3H、11C、13C、14C、15N、18F、31P、32P、35S、36Cl和125I等代替。除另有说明，当一个位置被特别地指定为氘(D)时，该位置应理解为具有大于氘的天然丰度(其为0.015％)至少3000倍的丰度的氘(即，至少45％的氘掺入)。
附图说明
图1：受试蛋白与人源IL-4Rα表达细胞的全细胞结合活性剂量-响应曲线
图2：受试蛋白对IL-4/IL-13信号通路阻断作用的剂量-响应曲线
图3：受试蛋白在TF-1细胞中的内吞率-时间曲线
图4：气管内给药后ADC-2和游离Budesonide在各组织的时间-浓度曲线
图5：测试例6肺泡灌洗液内白细胞与分型细胞计数
图6：测试例7肺泡灌洗液内白细胞与分型细胞计数
具体实施方式
以下结合实施例进一步描述本公开所述的化合物、可药用盐的制备，但这些实施例并非限制本公开中的范围。
本公开中实施例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
NMR位移(δ)以10-6(ppm)的单位给出。NMR的测定是用Bruker AVANCE-400核磁仪，测定溶剂为氘代二甲基亚砜(DMSO-d6)，氘代氯仿(CDCl3)，氘代甲醇(CD3OD)，内标为四甲基硅烷(TMS)。
MS的测定用Shimadzu 2010 Mass Spectrometer或Agilent 6110AMSD质谱仪。
HPLC的测定使用Shimadzu LC-20A systems、Shimadzu LC-2010HT series或安捷伦Agilent 1200 LC高压液相色谱仪(Ultimate XB-C18 3.0*150mm色谱柱或Xtimate C18 2.1*30mm色谱柱)。
手性HPLC分析测定使用Chiralpak IC-3 100×4.6mm I.D.,3um、Chiralpak AD-3 150×4.6mm I.D.,3um、Chiralpak AD-3 50×4.6mm I.D.,3um、Chiralpak AS-3 150×4.6mm I.D.,3um、Chiralpak AS-3 100×4.6mm I.D.,3μm、ChiralCel OD-3 150×4.6mm I.D.,3um、Chiralcel OD-3 100×4.6mm I.D.,3μm、ChiralCel OJ-H 150×4.6mm I.D.,5um、Chiralcel OJ-3 150×4.6mm I.D.,3um色谱柱；薄层层析硅胶板使用烟台黄海HSGF254或青岛GF254硅胶板，薄层色谱法(TLC)使用的硅胶板采用的规格是0.15mm～0.2mm，薄层层析分离纯化产品采用的规格是0.4mm～0.5mm。
柱层析一般使用烟台黄海硅胶100～200目、200～300目或300～400目硅胶为载体。
手性制备柱使用DAICEL CHIRALPAK IC(250mm*30mm,10um)或Phenomenex-Amylose-1(250mm*30mm,5um)。
CombiFlash快速制备仪使用Combiflash Rf150(TELEDYNE ISCO)。
激酶平均抑制率及IC50值的测定用NovoStar酶标仪(德国BMG公司)。
本公开的已知的起始原料可以采用或按照本领域已知的方法来合成，或可购买自ABCRGmbH&Co.KG，Acros Organics，Aldrich Chemical Company，韶远化学科技(Accela ChemBio Inc)、达瑞化学品等公司。
实施例中无特殊说明，反应能够均在氩气氛或氮气氛下进行。
氩气氛或氮气氛是指反应瓶连接一个约1L容积的氩气或氮气气球。
氢气氛是指反应瓶连接一个约1L容积的氢气气球。
加压氢化反应使用Parr 3916EKX型氢化仪和清蓝QL-500型氢气发生器或HC2-SS型氢 化仪。
氢化反应通常抽真空，充入氢气，反复操作3次。
微波反应使用CEM Discover-S 908860型微波反应器。
实施例中无特殊说明，溶液是指水溶液。
实施例中无特殊说明，反应的温度为室温，为20℃～30℃。
实施例中的反应进程的监测采用薄层色谱法(TLC)，反应所使用的展开剂，纯化化合物采用的柱层析的洗脱剂的体系和薄层色谱法的展开剂体系包括：A：二氯甲烷/甲醇体系，B：正己烷/乙酸乙酯体系，C：石油醚/乙酸乙酯体系，D：石油醚/乙酸乙酯/甲醇，溶剂的体积比根据化合物的极性不同而进行调节，也可以加入少量的三乙胺和醋酸等碱性或酸性试剂进行调节。
1.0 M Tris buffer pH＝8.30±0.1的配制：
50mL容量瓶中称取tris 6.0g，加入40mL纯化水，振荡溶解，滴加入浓盐酸1.2mL，调pH至8.30，再用纯化水定容。
Buffer A的配制：
在2.0L的容器中，加入KH2PO4(8.50g)、K2HPO4(8.56g)、NaCl(5.86g)和EDTA(1.50g)，加入1.6L注射用水，搅拌半小时，完全溶解后再用注射用水定容至2.0L，测定pH为6.30±0.1。
下述实验中所用缩写代表的含义如下:
DAST:二乙胺基三氟化硫；THF：四氢呋喃；NMP:N-甲基吡咯烷酮；DCM:二氯甲烷；m-CPBA:间氯过氧苯甲酸；DIEA:N,N-二异丙基乙胺；TEA：三乙胺；Boc：叔丁氧羰基，MeOH:甲醇；Et2O:乙醚。
本申请中25G7、7B10抗体的制备、纯化方法已在申请号为WO2020038454A1专利文件中记载，前述申请文件的全部内容均引入本公开。
实施例1：N-((10S)-10-苄基-1-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-羟基-6a,8a-二甲基-4-氧代-10-丙基-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-十二氢-8bH-萘并[2',1':4,5]茚并[1,2-d][1,3]二氧杂环戊-8b-基)-1,6,9,12,15-五氧-3-氧代-5,8,11,14-四聚乙二醇-16-基)-1-(2-溴乙酰氨基)-3,6,9,12-四氧代十五烷-15-酰胺(化合物1)的制备

步骤1)(((9H-芴-9-基)甲氧基)羰基)甘氨酰甘氨酰-L-苯丙氨酸叔丁基酯(化合物1b)的制备
将化合物1a(5.14g，20.0mmol，1.0eq)和L-苯丙氨酸叔丁酯盐酸盐(7.08g，20.0mmol，1.0eq)置入250mL三口烧瓶中，加入无水DMF(60mL)溶清。冰浴冷却，再加入HATU(9.12g,24.0mmol,1.2eq)和DIEA(7.74g，60.0mmol，3.0eq)，保持冰浴反应2小时。向反应液中加入水，乙酸乙酯萃取，有机相用饱和食盐水洗涤，干燥，减压浓缩得化合物1b(11.5g,100％yield)，粗品可直接投入下一步。
MS(ESI):m/z 580.3[M+Na]+。
1H NMR(400MHz,DMSO-d6)δ8.21-8.16(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).
步骤2)甘氨酰甘氨酰-L-苯丙氨酸叔丁酯(化合物1c)的制备
将化合物1b(5.57g，10.0mmol，1.0eq)置入250mL三口烧瓶中，加入无水DCM(60mL)溶清，冰浴冷却。缓慢加入哌啶(8.5g，100.0mmol，10.0eq)，加毕之后维持室温搅拌反应约2小时。直接将反应液浓缩至干，粗品经柱层析纯化得化合物1c(2.70g，81％yield)
MS(ESI):m/z 358.3[M+H]+。
步骤3)(1-(9H-芴-9-基)-3-氧代-2,7,10,13,16-五氧-4-四聚乙二醇-19-氧基)甘氨酰甘氨酰-L-苯丙氨酸叔丁酯(化合物1d)的制备
100mL三口烧瓶中加入原料1-(9H-芴-9-基)-3-氧代-2,7,10,13,16-五氧-4-四聚乙二醇-19-油酸(1.0g，2.05mmol，1.0eq；来源于通莱生化)和1c(0.69g，2.05mmol，1.0eq)，加入无水DMF(25mL)溶清，冰浴冷却。加入HATU(1.01g,2.67mmol,1.3eq)和DIEA(529mg，4.10mmol，2.0eq)，维持冰浴反应1小时。向反应液中加入水，乙酸乙酯萃取，饱和食盐水洗涤，干燥且减压浓缩得化合物1d(1.60g,97％yield)。
MS(ESI):m/z 805.3[M+H]+。
步骤4)(1-(9H-芴-9-基)-3-氧代-2,7,10,13,16-五氧-4-四聚乙二醇-19-氧基)甘氨酰甘氨酰-L-苯丙氨酸(化合物1e)的制备
100mL单口瓶中加入化合物1d(1.60g，1.99mmol，1.0eq)，加入无水二氯甲烷(16mL)溶解。室温下加入三氟乙酸(8mL)，维持搅拌反应2小时。将反应液直接浓缩至干，加入乙酸乙酯溶解，饱和食盐水洗涤，干燥，减压浓缩得化合物1e(1.23g，83％yield)；
MS(ESI):m/z 749.3[M+H]+。
步骤5)(9H-芴-9-基)甲基(2-(((2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-羟基-6a,8a-二甲基-4-氧代-10-丙基-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-十二氢-8bH-萘并[2',1':4,5]茚并[1,2-d][1,3]二氧杂环戊-8b-基)-2-氧代乙氧基)甲基)氨基)-2-氧代乙基)氨基甲酸酯(化合物1f)的制备
100mL三口瓶中加入原料布地奈德(1.29g，3.0mmol，1.0eq)和(2-((((9H-芴-9-基)甲氧基)羰基)氨基)乙酰氨基)乙酸甲酯(1.16g，3.15mmol，1.0eq；参考文献“Tetrahedron,2018,74(15),1951–1956”的方法制备)及对甲苯磺酸吡啶盐(75mg，0.30mmol，0.1eq)。室温下加入加入无水四氢呋喃(20mL)，回流加热反应4小时。直接将反应液浓缩至干，粗品柱层析纯化得1f(0.54g，24％yield)。
MS(ESI):m/z 739.4[M+H]+。
1H NMR(400MHz,DMSO-d6)δ8.76-8.69(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＝7.5Hz,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).
步骤6)2-氨基-N-((2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-羟基-6a,8a-二甲基-4-氧代-10-丙基-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-十二氢-8bH-萘并[2',1':4,5]茚并[1,2-d][1,3]二氧杂环戊-8b-基)-2-氧代乙氧基)甲基)乙酰胺(化合物1g)的制备
25mL单口瓶中置入化合物1f(540mg，0.73mmol，1.0eq)，加入无水二氯甲烷(10mL) 溶解。冰浴冷却，加入DBU(167mg，1.10mmol，1.5eq)，维持搅拌反应30分钟。加入水分液，水相用二氯甲烷萃取两次，饱和食盐水洗涤，减压浓缩得化合物1g(450mg，100％yield)，粗品可以直接用于下一步反应。
MS(ESI):m/z 517.4[M+H]+。
步骤7)(9H-芴-9-基)甲基((10S)-10-苄基-1-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-羟基-6a,8a-二甲基-4-氧代-10-丙基-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-十二氢-8bH-萘并[2',1':4,5]茚并[1,2-d][1,3]二氧杂环戊-8b-基)-1,6,9,12,15,18-六氧代-3,21,24,27,30-五氧代-5,8,11,14,17-五氨杂三十烷-32-基)氨基甲酸酯(化合物1h)的制备
100mL三口烧瓶中置入化合物1g(0.45g粗品，折算为0.73mmol，1.0eq)和1e(0.55g，0.73mmol，1.0eq)，加入无水DMF(9mL)溶清。冰浴冷却，再依次加入HATU(361mg,0.95mmol,1.3eq)和DIEA(189mg，1.46mmol，2.0eq)，维持搅拌反应1小时。向反应液中加入水，二氯甲烷萃取两次，合并有机相，饱和食盐水洗涤一次，干燥且减压浓缩得粗品。将粗品在混合溶剂石油醚和乙酸乙酯中打浆纯化，得化合物1h(710mg，78％yield)。
MS(ESI):m/z 1269.7[M+Na]+。
步骤8)1-氨基-N-((10S)-10-苄基-1-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-羟基-6a,8a-二甲基-4-氧代-10-丙基-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-十二氢-8bH-萘并[2',1':4,5]茚并[1,2-d][1,3]二氧杂环戊-8b-基)-1,6,9,12,15-五氧代-3-氧代-5,8,11,14-聚四乙二醇-16-基-3,6,9,12-四氧代十五烷-15-酰胺(化合物1i)的制备
25mL单口瓶中置入化合物1h(350mg，0.28mmol，1.0eq)，加入无水二氯甲烷(10mL)溶清。冰浴冷却，加入DBU(64mg，0.42mmol，1.5eq)，维持搅拌反应1小时。直接将反应液浓缩至干，得粗品，再经柱层析纯化得化合物1i(289mg，89％yield)。
MS(ESI):m/z 1025.6[M+H]+。
步骤9)N-((10S)-10-苄基-1-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-羟基-6a,8a-二甲基-4-氧代-10-丙基-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-十二氢-8bH-萘并[2',1':4,5]茚并[1,2-d][1,3]二氧杂环戊-8b-基)-1,6,9,12,15-五氧-3-氧代-5,8,11,14-四聚乙二醇-16-基)-1-(2-溴乙酰氨基)-3,6,9,12-四氧代十五烷-15-酰胺(化合物1)的制备
25ml Schlenk-Tube中置入溴乙酸固体(35.2mg，0.127mmol，1.0eq)和EEDQ(63mg，0.25mmol，2.0eq)，加入无水DMF(5mL)溶清。维持室温搅拌反应1小时。化合物1i(130mg，0.127mmol，1.0eq)溶解在DMF(2mL)中，将此溶液逐滴加入到上述制备的活性酯溶液中，加毕再维持室温反应2小时。向反应体系中加入水，乙酸乙酯萃取两次，合并有机相，饱和食盐水洗涤，干燥且减压浓缩得粗品，再经柱层析纯化得化合物1(39mg,27％yield)。
MS(ESI):m/z 1145.5/1147.6[M+1]+。
1H NMR(400MHz,CDCl3)δ8.62-8.57(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＝9.6Hz,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).
实施例2：
N-((10S)-10-苄基-1-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-羟基-6a,8a-二甲基-4-氧代-10-丙基l-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-十二氢-8bH-萘并[2',1':4,5]茚并[1,2-d][1,3]二氧杂环戊-8b-基)-1,6,9,12,15-五氧-3-氧代-5,8,11,14-四聚乙二醇-16-基)-1-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)-3,6,9,12-四氧代十五烷-15-酰胺
步骤1)(((9H-芴-9-基)甲氧基)羰基)甘氨酰甘氨酰-L-苯丙氨酸(化合物2a)的制备
100mL单口瓶中置入化合物1b(1.50g，2.69mmol，1.0eq)，加入4M HCl的1,4-二氧六环溶液(20mL)溶解。室温下维持搅拌4小时至原料1b反应完全。将反应液直接浓缩至干，得化合物1a(1.34g，100％yield)。
MS(ESI):m/z 502.3[M+1]+
步骤2)(9H-芴-9-基)甲基-((10S)-10-苄基-1-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-羟基-6a,8a-二甲基-4-氧代-10-丙基-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-十二氢-8bH-萘并[2',1':4,5]茚并[1,2-d][1,3]二氧杂环戊-8b-基)-1,6,9,12,15-五氧代-3-氧-5,8,11,14-四聚乙二醇-16-基)氨基甲酸酯(化合物2b)的制备
将化合物2a(0.97g，1.63mmol，1.0eq)和1g(1.0g，1.95mmol，1.2eq)置于100mL三口烧瓶中。加入DMF(20mL)，冰浴冷却。依次加入HATU(927mg，2.44mmol， 1.5eq)和DIEA(420mg，3.26mmol，2.0eq)，加完后，维持冰浴反应1-2小时。将反应液浓缩，经柱层析纯化得化合物2b(860mg，53％yield)。
MS(ESI):m/z 1000.5[M+1]+
步骤3(2S)-2-(2-(2-氨乙酰氨基)乙酰氨基-N-(2-(((2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-羟基-6a,8a-二甲基-4-氧代-10-丙基-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-十二氢-8bH-萘并[2',1':4,5]茚并[1,2-d][1,3]二氧杂环戊-8b-基)-2-氧代乙氧基)甲基)氨基)-2-氧代乙基)-3-苯丙酰胺(化合物2c)的制备
25mL单口瓶中置入化合物2b(860mg，0.86mmol，1.0eq)，加入无水DMF烷(10mL)溶清。冰浴冷却，加入DBU(196mg，1.29mmol，1.5eq)，维持搅拌反应30分钟。直接将反应液浓缩，经柱层析纯化得化合物2c(485mg，72％yield)。
MS(ESI):m/z 778.4[M+1]+
步骤4)N-((10S)-10-苄基-1-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-羟基-6a,8a-二甲基-4-氧代-10-丙基l-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-十二氢-8bH-萘并[2',1':4,5]茚并[1,2-d][1,3]二氧杂环戊-8b-基)-1,6,9,12,15-五氧-3-氧代-5,8,11,14-四聚乙二醇-16-基)-1-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)-3,6,9,12-四氧代十五烷-15-酰胺(化合物2d)的制备
100mL三口烧瓶中置入化合物2c(480mg，0.62mmol，1.0eq)和1-(2,5-二氧基-2,5-二氢-1H-吡咯-1-基)-3,6,9,12-四氧戊二酸-15-油酸(213mg，0.62mmol，1.0eq；来源于通莱生化)，加入无水DMF(10mL)溶清。冰浴冷却，再依次加入HATU(352mg,0.93mmol,1.5eq)和DIEA(159mg，1.23mmol，2.0eq)，维持搅拌反应1小时。直接将反应液浓缩，经柱层析纯化得化合物2d(360mg，53％yield)。
MS(ESI):m/z 1105.6[M+1]+
1H NMR(400MHz,DMSO-d6)δ8.61-8.56(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).
实施例3：25G7-Fab蛋白制备流程
合成抗体的基因序列，亚克隆至pcDNA3.4载体。连接产物转化Top10感受态细胞，挑取阳性克隆扩大培养。克隆接种于培养基中扩大培养，大量抽提含25G7-Fab抗体的脱氧核糖核酸序列的质粒。采用脂质体转染，将表达质粒与转染试剂混合后，加入Expi 293细胞中，恒温培养箱培养5天后收获细胞培养物。将细胞培养物离心后取上清，过滤除去细胞碎片，收集澄清液。使用kappa select层析柱捕获目的蛋白。目的蛋白经过滤，用Nanodrop测定A280吸光度值并除以消光系数的方法得到蛋白浓度，乘以体积以确定最终产量，凝胶电泳、体积排阻色谱法对蛋白进行SDS-PAGE、SEC-HPLC及内毒素检测，得到最终产物是25G7-Fab。
实施例4：AZD1402-TAG蛋白制备流程
AZD1402-TAG(蛋白序列来自于专利WO2020200960A1中SEQ ID NO1，为了便于蛋白 制备纯化，该序列C端融合了His tag，最终命名为AZD1402-TAG)。
AZD1402-TAG
合成上述蛋白的编码基因序列，亚克隆至pcDNA3.4。将表达质粒与转染试剂混合后37℃孵育15分钟，将混合液逐滴加入HEK293细胞液中，细胞液37℃摇床培养一周后，将细胞培养物离心后取上清。使用不含咪唑的缓冲液平衡镍亲和层析柱，然后将蛋白样品流经镍亲和层析柱进行上样，再次使用不含咪唑的缓冲液平衡后，用含有高浓度咪唑的缓冲液洗脱柱结合蛋白。并将洗脱蛋白转移至透析袋于1×PBS中透析，置换为PBS存储缓冲液。经检测，获得目的蛋白。
实施例5：抗体药物偶联物ADC-1的制备
Buffer A的配制：在2.0L的容器中，加入KH2PO4(8.50g)、K2HPO4(8.56g)、NaCl(5.86g)和EDTA(1.50g)，加入1.6L注射用水，搅拌半小时，完全溶解后再用注射用水定容至2.0L，测定pH为6.30±0.1。
于37℃，向抗体25G7-Fab(重链序列如SEQ ID NO：50所示，轻链序列如SEQ ID NO：45所示)的buffer A缓冲水溶液(pH＝6.3的0.05M缓冲水溶液；10.0mg/ml，0.5mL，0.125mmol)加入配制好的三(2-羧乙基)膦(TCEP)的水溶液(2.5mM，100.0uL，0.250mmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将1.0 M Tris buffer(50uL)加入至上述反应液中，再将化合物1(1.43mg，1.250mmol)溶解于25ul DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：buffer A)，并用超滤管浓缩得到标题产物ADC-1的buffer A缓冲液(6.06mg/mL，0.60mL)，于4℃冷冻储存。
实施例6：抗体药物偶联物ADC-2的制备
于37℃，向抗体25G7-Fab的buffer A缓冲水溶液(pH＝6.3的0.05M缓冲水溶液；10.0mg/ml，6.0mL，1.50mmol)加入配制好的三(2-羧乙基)膦(TCEP)的水溶液(10.0mM，1.50mL，15.0mmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物2(12.2mg，12.0mmol)溶解于300ul DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：buffer A)，并用超滤管浓缩得到标题产物ADC-2的buffer A缓冲液(3.65mg/mL，12.8mL)，于4℃冷冻储存。
生物学评价
以下结合测试例进一步描述解释本公开中，但这些实施例并非意味着限制本公开中的范围。
测试例1：抗体药物偶联物ADC-2与人源IL-4Rα蛋白的亲和力检测
1、受试蛋白
抗体药物偶联物ADC-2，25G7-Fab，AZD1402-TAG
2、实验方法
采用表面等离子共振技术(surface plasmon resonance,SPR)检测抗体药物偶联物ADC-2、25G7-Fab及AZD1402-TAG与人源IL-4Rα抗原蛋白的亲和力。Biacore 8K(Cytiva)中放入SA传感器芯片(Cytiva)，Biotin标记的人源IL-4Rα蛋白(SinoBiological，CAT#10402-H08H-B)捕获于SA传感器芯片上，流动相采用HBS-EP+缓冲溶液(10mM HEPES,150mM NaCl,3mM EDTA,0.05％surfactant P20)。各受试蛋白用HBS-EP+缓冲溶液进行2倍梯度稀释后作为分析物进样，设置流速为30μL/min，ADC-2、25G7-Fab和AZD1402-TAG的检测时间为结合100秒，解离600秒。采用Biacore 8K evaluation software软件(Cytiva)进行分析，获得各受试蛋白的亲和力数据。
3、实验结果
表1受试蛋白与人源IL-4Rα蛋白结合的亲和力KD值
结果如表1所示，ADC-2结合人源IL-4Rα抗原蛋白的KD值为11.0pM，略优于AZD1402-TAG和25G7-Fab。
测试例2：抗体药物偶联物ADC-2与人源IL-4Rα表达细胞的全细胞结合活性
1、受试蛋白
抗体药物偶联物ADC-2，25G7-Fab，hu25G7(重链序列如SEQ ID NO：44所示，轻链序列如SEQ ID NO：45所示)，AZD1402-TAG。
2、实验方法
TF-1及Karpas 299细胞株经鉴定可表达IL-4Rα。将处于对数生长期的TF-1和Karpas299细胞收集后，计数并调整细胞密度，以1×105细胞/孔接入圆底96孔板。将细胞与不同浓度的抗IL-4R抗体(25G7、裸抗25G7-Fab)、抗体药物偶联物ADC-2及AZD1402-TAG在4℃条件下孵育45分钟，用FACS Buffer(含2％FBS的PBS)洗去抗体后，加入荧光标记二抗进行染色，25G7-Fab、hu25G7及ADC-2采用FITC偶联的抗人IgG(Fab特异性)二抗(Sigma，CAT#F5512-1ML)，AZD1402-TAG采用FITC偶联的抗His抗体(GenScript，CAT#A01620)。将细胞与二抗在4℃条件下孵育30分钟，随后用FACS Buffer洗涤两次，流式细胞仪进行检测(BD，FACS Celesta)，检测结果采用FlowJo(FlowJo,LLC)软件进行分析。
3、实验结果
表2受试蛋白与人源IL-4Rα表达细胞的全细胞结合活性EC50值
结果如图1及表2所示，ADC-2结合TF-1细胞的EC50值为0.107nM，与裸抗25G7-Fab和hu25G7相当。AZD1402-TAG与TF-1细胞的结合作用强，但因检测抗体不同，数据与ADC-2、25G7-Fab等不具有可比性。
测试例3：抗体药物偶联物ADC-2对IL-4/IL-13信号通路的阻断作用
1、受试蛋白
抗体药物偶联物ADC-2，25G7-Fab。
2、实验方法
STAT6的活化是IL-4/IL-13信号通路激活的关键步骤。本实施例中，ADC-2对IL-4/IL-13信号通路的阻断作用通过HEK-BlueTMIL-4/IL-13报告基因细胞株进行评估。该细胞购自Invivogen(Cat#hkb-il413)，其中过表达了人源STAT6基因及由磷酸化STAT6诱导表达的分泌型碱性磷酸酶报告基因(Secreted alkaline phos-phatase，SEAP)，可通过 SEAP底物QUANTI-Blue检测细胞培养上清中分泌的SEAP的含量以评估IL-4/IL-13信号通路的活化水平。
将处于对数生长期的HEK-BlueTMIL-4/IL-13细胞收集后调整密度为5×105细胞/ml，每孔100μL加入96孔平底培养板中，于37℃，5％CO2条件下培养24小时，而后每孔加入20μL梯度稀释的受试蛋白和20μL重组人IL-4或IL-13，将细胞培养板放在37℃，5％CO2培养箱中继续孵育20～24小时。培养结束后，取出细胞培养板，每孔吸取20μL细胞培养上清转移至另一块96孔板，加入37℃预热的QUANTI-Blue显色液180μL/孔，在37℃孵育1小时后，检测620nm波长下的光吸收值。
3、实验结果
表3受试蛋白对IL-4/IL-13信号通路阻断作用的IC50值
结果如图2及表3所示，各受试蛋白对重组人IL-4和IL-13诱导的STAT6的活化均有阻断作用，ADC-2阻断IL-4信号通路的IC50值为0.68nM，与25G7-Fab的IC50值0.61nM接近；ADC-2阻断IL-13信号通路的IC50值为10.60nM，与25G7-Fab的IC50值14.59nM相当。
测试例4：抗体药物偶联物ADC-2在TF-1细胞中的内吞活性
1、受试蛋白
抗体药物偶联物ADC-2，25G7-Fab，hu25G7，AZD1402-TAG。
2、实验方法
将生长状态良好的TF-1细胞收集后调整密度为1×106细胞/mL，100μL/孔加入96孔细胞培养板中并置于4℃，加入终浓度为2nM的各受试蛋白，于4℃孵育1小时后放入细胞培养箱于4℃分钟，5％CO2条件下继续培养，每个时间点单点设置一块培养板，期间按不同孵育时间取出培养板，FACS buffer清洗并离心，随后加入荧光标记二抗进行孵育。25G7-Fab、hu25G7及ADC-2采用FITC偶联的抗人IgG(Fab特异性)二抗进行标记，AZD1402-TAG采用FITC偶联的抗His抗体进行标记。将细胞与二抗在4℃条件下孵育30分钟，随后用FACS Buffer洗涤两次，流式细胞仪进行检测(BD，FACS Celesta)，检测结果采用FlowJo(FlowJo,LLC)软件进行分析。特定时间点的内吞率计算公式为：(gMFITest Ab at time X-gMFIISO Ab at time X)/(gMFITest Ab at time zero-gMFIISO Ab at time zero)*100％
3、实验结果
实验结果如图3所示，各受试蛋白均可检测到其在TF-1细胞中的内吞信号。ADC-2和hu25G7的内吞作用活性相当，3小时内吞率在70％左右；25G7-Fab的内吞作用更快， 内吞率也更高。相比于AZD1402-TAG，ADC-2、25G7-Fab和hu25G7的内吞速率和内吞率均显著优于AZD1402-TAG约在2小时左右达到平台，ADC-2的内吞活性比25G7-Fab稍弱。
测试例5：抗体药物偶联物ADC-2在小鼠模型上的药代动力学测试
1、受试蛋白
抗体药物偶联物ADC-2
2、实验方法
2.1实验动物
C57BL6/J小鼠，8周龄，雄性，赛业(苏州)生物科技有限公司
2.2小鼠给药操作
小鼠接受气管内雾化给药(intratracheal，i.t.)，于给药后的不同时间点进行下颌采血300-400μL，静置2小时后，7500rpm，4℃离心10min，采集血清。采血后将小鼠进行麻醉并固定，将小鼠颈部肌肉进行钝性分离，暴露出气管，将气管横向开口，缓慢插入灌洗针，将预先抽装好的4℃预冷生理盐水缓慢推入肺组织进行灌洗，并收集肺泡灌洗液(BALF)。一共灌洗3次，前两次分别用300μL生理盐水灌洗，第3次用400μL灌洗。收集好的肺泡灌洗液1000rpm，4℃离心10min，采集上清液。安乐死小鼠后，剖开小鼠胸腔，分离并收集完整小鼠肺组织，用PBS冲洗掉肺组织表面的血渍，纸巾蘸干表面PBS后，对肺组织称重。将肺组织置于2mL离心管中，加入1mL细胞裂解液(CST，#9803)，匀浆器(Shanghai Jing Xin，Tissuelyser-48)60Hz研磨2min后，10,000×g，4℃离心10min，上清液即为肺组织匀浆液，检测匀浆液蛋白浓度(Pierce,23227)。血清、BALF和肺组织匀浆液置于-80℃保存。给药方案见表4，采样方案见表5。
表4给药方案
表5采样方案
2.3 ADC-2含量检测
(1)包被：用碳酸盐缓冲液稀释抗原hIL-4Rα至1μg/mL,(Acro，ILR-H5221)，向 ELISA板中每孔加入100μL，4℃过夜孵育(16-18h)。
(2)洗板：ELISA板恢复至室温，每孔加入300μL 0.05％PBST(PBS+0.05％Tween20)缓冲液洗板，共洗板3次。
(3)封闭：每孔加入300μL含3％BSA的PBS，400rpm室温摇板孵育2h。
(4)洗板：每孔加入300μL 0.05％PBST，洗板3次。
(5)样品孵育：将100μL稀释后的血清、BALF或肺组织匀浆液加入ELISA板中，400rpm室温摇板孵育1.5h。
(6)洗板：每孔加入300μL 0.05％PBST，洗板3次。
(7)检测：用Assay buffer(含0.5％BSA的0.05％PBST)将检测抗体(Invitrogen，A18811)稀释10000倍后加入ELISA板中，每孔100μL，400rpm室温摇板孵育1h。
(8)洗板：每孔加入300μL 0.05％PBST，洗板3次。
(9)显色：每孔加入100μL TMB底物(Sera care，5120-0080)，室温孵育15min。
(10)终止：每孔加入100μL底物反应终止液(Solarbio，C1058)，检测620nm波长下的光吸收值。
2.4游离Budesonide含量检测
取30μL血清、BALF或肺组织匀浆液，加入30.0μL内标工作溶液(5.00ng/mL布地奈德-D8)和120μL乙腈，涡流1min，13,000rpm，4℃离心5min，取上清100μL至96孔板中，加入100μL 0.3％FA稀释液，1,000rpm室温摇板孵育15min，取20μL进行LC-MS/MS分析(日本岛津，岛津液相色谱系统；AB Sciex，Triple Quad 6500+型三重四极杆串联质谱仪)。
3、实验结果
抗体药物偶联物ADC-2和游离Budesonide的药代动力学参数如表6所示，药时曲线如图4所示。
表6 ADC-2和游离Budesonide的PK参数
实验结果表明，抗体药物偶联物ADC-2气管内给药后，药物主要分布于血清和BALF， 血清暴露量低。各组织中Budesonide的暴露量均很低。
测试例6：ADC-2在OVA诱导的小鼠哮喘模型上的体内药效
1、受试物
抗体药物偶联物ADC-2，25G7-Fab，AZD1402-TAG，Budesonide。
2、实验方法
2.1实验动物
IL-4 hu/hu-IL-4Rαhu/hu双转基因C57BL6/J小鼠，8周龄，雌性，赛业(苏州)生
物科技有限公司
2.2小鼠给药操作
将动物随机分为正常对照动物和造模动物。于Day 0，7和14分别腹腔注射100μL含200μg OVA(Sigma，A5503)的PBS溶液，并使用等体积的氢氧化铝作为佐剂对小鼠进行致敏。于Day 21-27，小鼠每天吸入一次3％OVA的雾化溶液，每次持续35min。于Day 21开始，吸入OVA雾化溶液前半小时，小鼠接受气管内雾化给药。具体给药方案见表7。
在Day 28，小鼠安乐死后，将小鼠颈部肌肉进行钝性分离，暴露出气管，将气管横向开口，缓慢插入灌洗针，将预先抽装好的4℃预冷生理盐水缓慢推入肺组织进行灌洗，并收集肺泡灌洗液。一共灌洗3次，前两次分别用300μL生理盐水灌洗，第3次用400μL灌洗。1,000rpm，4℃离心10min，细胞沉淀用350μL预冷生理盐水重悬后，用全自动血液分析仪进行白细胞与分型细胞计数。
表7给药方案
2.3实验指标
BALF内白细胞与分型细胞的数量。
2.4统计学分析
除非特别说明，两组别细胞计数之间的比较采用one-way ANOVA检验，p<0.05定义为有统计学显著性差异。
3、实验结果
实验结果如图5所示。相比于造模组，在等摩尔剂量下，抗体药物偶联物ADC-2显著降低了BALF内白细胞、嗜酸性粒细胞和嗜中性粒细胞的数量，对单核细胞的数量也有一定的抑制，药效优于AZD1402-TAG，并明显优于25G7-Fab和Budesonide。ADC-2在药效上体现出了25G7-Fab和Budesonide的协同效应。
测试例7：ADC-2在OVA诱导的小鼠哮喘模型上体内药效的剂量探索
1、受试物
抗体药物偶联物ADC-2，AZD1402-TAG，Budesonide。
2、实验方法
2.1实验动物
IL-4 hu/hu-IL-4Rαhu/hu双转基因C57BL6/J小鼠，8周龄，雌性，赛业(苏州)生物科技有限公司
2.2小鼠给药操作
给药和BALF采样操作同测试例6。具体给药方案见表8。
2.3实验指标
BALF内白细胞与分型细胞的数量。
2.4统计学分析
除非特别说明，两组别细胞计数之间的比较采用one-way ANOVA检验，p<0.05定义为有统计学显著性差异。
3、实验结果
实验结果如图6所示。相比于造模组，10μg ADC-2就能将肺泡灌洗液内嗜酸性粒细胞的数量显著降低，与AZD1402-TAG相当，并略优于远高于临床等效使用剂量的布地奈德。此外，10μg ADC-2对白细胞数量的抑制能力也非常显著，对单核细胞和中性粒细胞的数量也有一定程度的抑制。这些结果表明，1/10摩尔剂量的ADC-2就能实现与 AZD1402-TAG相当的药效。

Claims (21)

  1. 式(I)所示的抗体-药物偶联物，
    Ab-(L-D)k
    (I)
    其中，Ab为抗IL-4R抗体或其抗原结合片段，
    L为将Ab共价连接于D的连接子，且k为1至20，
    D为糖皮质激素或其残基，所述糖皮质激素选自布地奈德或环索奈德。
  2. 根据权利要求1所述的抗体-药物偶联物，其中D为：
  3. 根据权利要求1或2所述的抗体-药物偶联物，其中所述抗IL-4R的抗体或其抗原结合片段包含选自以下(I)至(IV)中的任一项：
    (I)重链可变区，其包含分别如SEQ ID NO：3、SEQ ID NO：4和SEQ ID NO：5所示的HCDR1、HCDR2和HCDR3；和
    轻链可变区，其包含分别如SEQ ID NO：38、SEQ ID NO：7和SEQ ID NO：40所示的LCDR1、LCDR2和LCDR3；
    (II)重链可变区，其包含分别如SEQ ID NO：11、SEQ ID NO：12和SEQ ID NO：13所示的HCDR1、HCDR2和HCDR3；和
    轻链可变区，其包含分别如SEQ ID NO：14、SEQ ID NO：15和SEQ ID NO：16所示的LCDR1、LCDR2和LCDR3；
    (III)重链可变区，其包含分别如SEQ ID NO：3、SEQ ID NO：4和SEQ ID NO：5所示的HCDR1、HCDR2和HCDR3；和
    轻链可变区，其包含分别如SEQ ID NO：6、SEQ ID NO：7和SEQ ID NO：8所示的LCDR1、LCDR2和LCDR3；
    (IV)重链可变区，其包含分别如SEQ ID NO：3、SEQ ID NO：4和SEQ ID NO：5所示的HCDR1、HCDR2和HCDR3；和
    轻链可变区，其包含分别如SEQ ID NO：42、SEQ ID NO：39和SEQ ID NO：8所示的LCDR1、LCDR2和LCDR3。
  4. 根据权利要求1-3任一项所述的抗体-药物偶联物，所述的抗IL-4R的抗体选自以下中的任一项：鼠源抗体、嵌合抗体、全人抗体和人源化抗体；
    优选地，所述的抗IL-4R的抗体或其抗原结合片段包含来源于人种系轻链IGKV3-11*01 的FR或与其至少有95％同一性的FR；所述FR优选包含回复突变，所述回复突变选自46P、47W和71Y中的一个或多个；和/或
    包含来源于人种系重链IGHV3-48*01的FR或与其至少有95％同一性的FR；所述FR优选包含回复突变，所述回复突变选自49A、67S和93T中的一个或多个；
    或者优选地，所述的抗IL-4R的抗体或其抗原结合片段包含来源于人种系轻链IGKV2D-29*01的FR或与其至少有95％同一性的FR；所述FR优选包含回复突变，所述回复突变选自4L和/或58I；和/或
    包含来源于人种系重链IGHV1-2*02的FR或与其至少有95％同一性的FR；所述FR优选包含回复突变，所述回复突变选自69L、71I、73K和94K中的一个或多个。
  5. 根据权利要求1-4任一项所述的抗体-药物偶联物，其中：所述的抗IL-4R的抗体或其抗原结合片段包含选自以下(i)至(vi)中的任一项：
    (i)重链可变区，其包含如SEQ ID NO：43所示的氨基酸序列或与SEQ ID NO：43具有至少90％、95％、98％、99％同一性的氨基酸序列；和
    轻链可变区，其包含如SEQ ID NO：37所示的氨基酸序列或与SEQ ID NO：37具有至少90％、95％、98％、99％同一性的氨基酸序列；
    (ii)重链可变区，其包含如SEQ ID NO：9所示的氨基酸序列或与SEQ ID NO：9具有至少90％、95％、98％、99％同一性的氨基酸序列；和
    轻链可变区，其包含如SEQ ID NO：10所示的氨基酸序列或与SEQ ID NO：10具有至少90％、95％、98％、99％同一性的氨基酸序列；
    (iii)重链可变区，其包含如SEQ ID NO：1所示的氨基酸序列或与SEQ ID NO：1具有至少90％、95％、98％、99％同一性的氨基酸序列；和
    轻链可变区，其包含如SEQ ID NO：2所示的氨基酸序列或与SEQ ID NO：2具有至少90％、95％、98％、99％同一性的氨基酸序列；
    (iv)重链可变区，其包含如SEQ ID NO：43所示的氨基酸序列或与SEQ ID NO：43具有至少90％、95％、98％、99％同一性的氨基酸序列；和
    轻链可变区，其包含如SEQ ID NO：41所示的氨基酸序列或与SEQ ID NO：41具有至少90％、95％、98％、99％同一性的氨基酸序列；
    (v)重链可变区，其包含如SEQ ID NO：25-27之一所示的序列或与SEQ ID NO：25-27之一具有至少90％、95％、98％或99％同一性的序列；和
    轻链可变区，其包含如SEQ ID NO：28-30之一所示的序列或与SEQ ID NO：28-30之一具有至少90％、95％、98％或99％同一性的序列；
    (vi)重链可变区，其包含如SEQ ID NO：31-33之一所示的序列或与SEQ ID NO：31-33之一具有至少90％、95％、98％、或99％同一性的序列；和
    轻链可变区，其包含如SEQ ID NO：34-36之一所示的序列或与SEQ ID NO：34-36之一具有至少90％、95％、98％或99％同一性的序列；
    优选地，所述抗IL-4R的抗体或抗原结合片段包含选自以下(vii)至(xii)中的任一项：
    (vii)序列如SEQ ID NO：43所示的重链可变区，序列如SEQ ID NO：37所示的轻链可变区；
    (viii)序列如SEQ ID NO：9所示的重链可变区，序列如SEQ ID NO：10所示的轻链可变区；
    (ix)序列如SEQ ID NO：1所示的重链可变区，序列如SEQ ID NO：2所示的轻链可变区；
    (x)序列如SEQ ID NO：43所示的重链可变区，序列如SEQ ID NO：41所示的轻链可变区；
    (xi)序列如SEQ ID NO：25-27之一所示的重链可变区，序列如SEQ ID NO：28-30之一所示的轻链可变区；
    (xii)序列如SEQ ID NO：31-33之一所示的重链可变区，序列如SEQ ID NO：34-36之一所示的轻链可变区。
  6. 根据权利要求1-5任一项所述的抗体-药物偶联物，其中：所述抗IL-4R的抗体或其抗原结合片段包含重链恒定区，所述重链恒定区为人源IgG1、IgG2、IgG3或IgG4的重链恒定区或其变体；和/或
    所述抗原结合片段为Fab、Fv、scFv、Fab’或F(ab’)2。
  7. 根据权利要求1-6任一项所述的抗体-药物偶联物，其中：所述抗IL-4R抗体或其抗原结合片段包含选自(a)-(d)中的任一项：
    (a)重链，其包含如SEQ ID NO：44所示的氨基酸序列或与SEQ ID NO：44具有至少90％、95％、98％、或99％同一性的氨基酸序列；和
    轻链，其包含如SEQ ID NO：45所示的氨基酸序列或与SEQ ID NO：45具有至少90％、95％、98％或99％同一性的氨基酸序列；
    (b)重链，其包含如SEQ ID NO：19所示的氨基酸序列或与SEQ ID NO：19具有至少90％、95％、98％、或99％同一性的氨基酸序列；和
    轻链，其包含如SEQ ID NO：20所示的氨基酸序列或与SEQ ID NO：20具有至少90％、95％、98％或99％同一性的氨基酸序列；
    (c)重链，其包含如SEQ ID NO：17所示的氨基酸序列或与SEQ ID NO：17具有至少90％、95％、98％或99％同一性的氨基酸序列；和
    轻链，其包含如SEQ ID NO：18所示的氨基酸序列或与SEQ ID NO：18具有至少90％、95％、98％或99％同一性的氨基酸序列；
    (d)重链，其包含如SEQ ID NO：44所示的氨基酸序列或与SEQ ID NO：44具有至少90％、95％、98％、或99％同一性的氨基酸序列；和
    轻链，其包含如SEQ ID NO：46所示的氨基酸序列或与SEQ ID NO：46具有至少90％、95％、98％或99％同一性的氨基酸序列；
    更优选地，所述抗IL-4R抗体或其抗原结合片段包含选自(e)-(i)中的任一项：
    (e)序列如SEQ ID NO：44所示的重链，序列如SEQ ID NO：45所示的轻链；
    (f)序列如SEQ ID NO：47所示的重链，序列如SEQ ID NO：45所示的轻链；
    (g)序列如SEQ ID NO：17所示的重链，序列如SEQ ID NO：18所示的轻链；
    (h)序列如SEQ ID NO：19所示的重链，序列如SEQ ID NO：20所示的轻链；
    (i)序列如SEQ ID NO：44所示的重链，序列如SEQ ID NO：46所示的轻链。
  8. 根据权利要求1-6任一项所述的抗体-药物偶联物，其中：所述抗IL-4R抗体或其抗原结合片段包含选自(a)-(f)中的任一项：
    (a)重链，其包含如SEQ ID NO：48所示的氨基酸序列或与SEQ ID NO：48具有至少90％、95％、98％、或99％同一性的氨基酸序列；和
    轻链，其包含如SEQ ID NO：18所示的氨基酸序列或与SEQ ID NO：18具有至少90％、95％、98％或99％同一性的氨基酸序列；
    (b)重链，其包含如SEQ ID NO：49所示的氨基酸序列或与SEQ ID NO：49具有至少90％、95％、98％、或99％同一性的氨基酸序列；和
    轻链，其包含如SEQ ID NO：18所示的氨基酸序列或与SEQ ID NO：18具有至少90％、95％、98％或99％同一性的氨基酸序列；
    (c)重链，其包含如SEQ ID NO：50所示的氨基酸序列或与SEQ ID NO：50具有至少90％、95％、98％或99％同一性的氨基酸序列；和
    轻链，其包含如SEQ ID NO：45示的氨基酸序列或与SEQ ID NO：45具有至少90％、95％、98％或99％同一性的氨基酸序列；
    (d)重链，其包含如SEQ ID NO：50所示的氨基酸序列或与SEQ ID NO：50具有至少90％、95％、98％、或99％同一性的氨基酸序列；和
    轻链，其包含如SEQ ID NO：46所示的氨基酸序列或与SEQ ID NO：46具有至少90％、95％、98％或99％同一性的氨基酸序列；
    (e)重链，其包含如SEQ ID NO：51所示的氨基酸序列或与SEQ ID NO：51具有至少90％、95％、98％、或99％同一性的氨基酸序列；和
    轻链，其包含如SEQ ID NO：45所示的氨基酸序列或与SEQ ID NO：45具有至少90％、95％、98％或99％同一性的氨基酸序列；
    (f)重链，其包含如SEQ ID NO：51所示的氨基酸序列或与SEQ ID NO：51具有至少90％、95％、98％、或99％同一性的氨基酸序列；和
    轻链，其包含如SEQ ID NO：46所示的氨基酸序列或与SEQ ID NO：46具有至少90％、95％、98％或99％同一性的氨基酸序列；
    更优选地，所述抗IL-4R抗体或其抗原结合片段包含选自(h)-(m)中的任一项：
    (h)序列如SEQ ID NO：48所示的重链，序列如SEQ ID NO：18所示的轻链；
    (i)序列如SEQ ID NO：49所示的重链，序列如SEQ ID NO：18所示的轻链；
    (j)序列如SEQ ID NO：50所示的重链，序列如SEQ ID NO：45所示的轻链；
    (k)序列如SEQ ID NO：50所示的重链，序列如SEQ ID NO：46所示的轻链；
    (l)序列如SEQ ID NO：51所示的重链，序列如SEQ ID NO：45所示的轻链；
    (m)序列如SEQ ID NO：51所示的重链，序列如SEQ ID NO：46所示的轻链。
  9. 根据权利要求1-8任一项所述的抗体-药物偶联物，其中L-D是由下式表示的化学部分：
    -Str-Pep-Sp-D，
    其中，Str是与Ab共价连接的伸展基单元，
    Sp为间隔单元，
    Pep选自氨基酸单元、二硫化物部分、磺酰胺部分或以下非肽化学部分：
    其中，W是-NH-亚杂环烷基-或杂
    环烷基；Y是亚杂芳基、亚芳基、-C(O)C1-6亚烷基、C2-6亚烯基、C1-6亚烷基或-C1-6亚烷基-NH-；每个R16独立选自C1-6烷基、C2-6烯基、-(C1-6亚烷基)NHC(NH)NH2或-(C1-6亚烷基)NHC(O)NH2；R17和R18各自独立选自氢、C1-6烷基、C2-6烯基、芳基、杂芳基，或R17和R18一起可形成C3-6环烷基；R19和R20各自独立选自C1-6烷基、C2-6烯基、芳基、杂芳基、(C1-6烷基)OCH2-，或R19和R20一起可形成C3-6环烷基环。
  10. 根据权利要求9所述的抗体-药物偶联物，其中Str选自下式表示的化学部分：
    其中R21选自-W1-C(O)-、-C(O)-W1-C(O)-、
    -(CH2CH2O)p1C(O)-、-(CH2CH2O)p1CH2C(O)-、-(CH2CH2O)p1CH2CH2C(O)-，其中W1选自C1-6亚烷基、C1-6亚烷基-环烷基或1至8个原子的直链杂烷基，所述杂烷基包含1至3个选自N、O或S的杂原子，其中所述烷基、环烷基和直链杂烷基各自独立地任选被一个或多个选自卤素、氘、羟基、氰基、氨基、C1-6烷基、卤代C1-6烷基、氘代C1-6烷基、C1-6烷氧基和C3-6环烷基所取代，p1各自独立地为1至20的整数；
    L1选自-NR22-(CH2CH2O)p2CH2CH2C(O)-、-NR22-(CH2CH2O)p2CH2C(O)-、-S-(CH2)p2C(O)-、-(CH2)p2C(O)-或单键，优选单键；p2各自独立地为1至20的整数，R22选自氢原子、C1-6烷基、卤代C1-6烷基或氘代C1-6烷基。
  11. 根据权利要求10所述的抗体-药物偶联物，其中R21选自-C1-6亚烷基C(O)-、-(CH2-CH2O)2C(O)-、-(CH2-CH2O)2CH2C(O)-、-(CH2-CH2O)2CH2CH2C(O)-、-(CH2-CH2O)3C(O)-和-(CH2-CH2O)4C(O)-。
  12. 根据权利要求10或11所述的抗体-药物偶联物，其中所述的Pep选自缬氨酸-瓜氨酸、丙氨酸-丙氨酸-天冬酰胺、甘氨酸-甘氨酸-赖氨酸、缬氨酸-赖氨酸(Val-lys)、缬氨酸-丙氨酸、缬氨酸-苯丙氨酸或甘氨酸-甘氨酸-苯丙氨酸-甘氨酸。
  13. 根据权利要求10-12任一项所述的抗体-药物偶联物，其中Sp选自-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)-、
  14. 根据权利要求1-13任一项所述的抗体-药物偶联物，其中所述抗体-药物偶联物中连接子L包含：顺丁烯二酰亚胺-(PEG)4-CH2CH2C(O)-Gly-Gly-Phe-Gly、顺丁烯二酰亚胺-(PEG)2-Val-Cit、顺丁烯二酰亚胺-(PEG)6-Val-Cit、顺丁烯二酰亚胺-(PEG)8-Val-Cit、顺丁烯二酰亚胺-(PEG)4-CH2CH2C(O)-Val-lys、顺丁烯二酰亚胺-(CH2)5-Val-Cit、顺丁烯二酰亚胺-(CH2)5-Val-lys、顺丁烯二酰亚胺-(CH2)5-Gly-Gly-Phe-Gly、顺丁烯二酰亚胺-(PEG)4-CH2CH2C(O)-Gly-Gly-Phe-Gly、顺丁烯二酰亚胺-(PEG)2-Ala-Ala-Asn、顺丁烯二酰亚胺-(PEG)6-Ala-Ala-Asn、顺丁烯二酰亚胺-(PEG)8-Ala-Ala-Asn、顺丁烯二酰亚胺-(PEG)4-三唑-(PEG)3-磺酰胺、顺丁烯二酰亚胺-(PEG)2-CH2CH2C(O)-Val-lys、顺丁烯二酰亚胺-(PEG)4-三唑-(PEG)3-磺酰胺或Mal-(PEG)4-三唑-(PEG)3-二硫化物。
  15. 根据权利要求1所述的抗体-药物偶联物，其由下式表示：
    其中k选自1至10，p1
    选自2、4、6或8，p3、p4各自独立地选自0、1或2；或
    其中k选自1至10，p1
    选自2、4、6或8，p3、p4各自独立地选自0、1或2。
  16. 根据权利要求15所述的抗体-药物偶联物，其选自：
    其中k选自1至10。
  17. 一种式II-A所示化合物或其可药用盐，
    其中，p1选自2、4、6或8，p3、p4各自独立地选自0、1或2，D为布地奈德或环索奈德。
  18. 一种式II-B所示化合物或其可药用盐，
    其中X为卤素，优选溴，p1选自2、4、6或8，p3、p4各自独立地选自0、1或2，D为布地奈德或环索奈德。
  19. 一种药物组合物，包含如权利要求1-16任一项所述的抗体-药物偶联物以及药学上可接受的赋形剂。
  20. 一种可吸入的药物组合物，包括式(I)所示的抗体-药物偶联物以及药学上可接受的载体，
    Ab-(L-D)k
    (I)
    其中，Ab为抗IL-4R的抗体或其抗原结合片段，优选抗IL-4R的Fab片段，
    L为将Ab共价连接于D的连接子，且k为1至20，
    D为糖皮质激素或其残基。
  21. 权利要求1-16任一项所述的抗体-药物偶联物或权利要求19或20所述的药物组合物在制备用于治疗或预防免疫性疾病或病症的药物中的用途，所述疾病或病症优选：哮喘、鼻息肉、慢性鼻窦炎、过敏性皮肤病、嗜酸细胞性食管炎、慢性阻塞性肺病、过敏性鼻炎、关节炎、炎症性疾病、变应性反应、自体免疫淋巴组织增生性综合征、自体免疫性溶血性贫血、巴雷特食管、自体免疫葡萄膜炎、结核病和肾病，更优选哮喘或过敏性皮肤病。
PCT/CN2023/073057 2022-01-29 2023-01-19 糖皮质激素的药物偶联物 WO2023143351A1 (zh)

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