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WO2025026216A1 - 一种含有糖皮质激素的抗体药物偶联物的药物组合物 - Google Patents

一种含有糖皮质激素的抗体药物偶联物的药物组合物 Download PDF

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Publication number
WO2025026216A1
WO2025026216A1 PCT/CN2024/107802 CN2024107802W WO2025026216A1 WO 2025026216 A1 WO2025026216 A1 WO 2025026216A1 CN 2024107802 W CN2024107802 W CN 2024107802W WO 2025026216 A1 WO2025026216 A1 WO 2025026216A1
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Prior art keywords
pharmaceutical composition
antibody
composition according
seq
drug conjugate
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PCT/CN2024/107802
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English (en)
French (fr)
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WO2025026216A9 (zh
Inventor
周晓丹
李金宇
叶秀
孙琼
卢韵
杨建�
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江苏恒瑞医药股份有限公司
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Publication of WO2025026216A1 publication Critical patent/WO2025026216A1/zh
Publication of WO2025026216A9 publication Critical patent/WO2025026216A9/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

  • Interleukin-4 consists of 153 amino acids and has a molecular weight of approximately 17 kDa. Initially, IL-4 was discovered because it could 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 consisting of a hydrophobic core of 4 ⁇ helices. IL-4 is secreted by TH2 cells and participates in TH2-mediated immune responses. It 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 multiple similar functions. Both IL-4 and IL-13 can promote B cell proliferation and induce the conversion of IgM type to IgE in combination with CD40/CD40L co-stimulation.
  • IL-4 promotes mast cell aggregation, upregulates the expression of mast cell high-affinity IgE receptor and B cell low-affinity IgE receptor CD23 (Fc ⁇ RII), upregulates the expression of vascular endothelial cell adhesion molecule (VCAM-1), and promotes the migration of eosinophils, T lymphocytes, monocytes and basophils.
  • IL-4 can promote the differentiation of naive T cells into TH2.
  • IL-4 needs to bind to membrane receptors to exert its biological function.
  • Human interleukin receptor (IL-4R) is a heterodimer formed by two polypeptide chains, one of which is an ⁇ chain with a high affinity for IL-4. Since the IL-4R ⁇ chain plays a dominant role in the binding of IL-4 in the IL-4R complex, IL-4R ⁇ is often used to replace IL-4R in many scientific studies and reports.
  • IL-4R is expressed on a variety of cells, including 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.
  • type I receptors composed of IL-4R ⁇ and ⁇ c are mainly expressed.
  • IL-4 mainly exerts its effects through type II receptors composed of IL-4R ⁇ and IL-13R ⁇ 1.
  • Type II receptor is a common receptor for IL-4 and IL-13.
  • IL-13 binds to IL-13R ⁇ 1 to exert its function.
  • Both type I 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 transduce signals through the insulin receptor substrate family (IRS), ultimately activating PI3-K and NF- ⁇ B in the nucleus. Blocking IL-4R can inhibit the biological functions of both IL-4 and IL-13.
  • IRS insulin receptor substrate family
  • Atopic dermatitis also known as atopic dermatitis or genetic allergic dermatitis
  • AD is a common dermatological disease, more common in children and adolescents, and often complicated by certain genetic allergic diseases such as allergic rhinitis and asthma.
  • TH2 factors IL-4, IL-5, IL- are related to the progression of AD.
  • Mice that overexpress TH2 factors such as IL-4 and IL-13 show skin protection defects and AD-like symptoms [14][15] . Elevated levels of IL-4 and IL-13 in AD patients hinder epidermal differentiation and the production of antimicrobial peptides.
  • IL-4-deficient mice reduce the occurrence of skin allergic inflammation.
  • IL-13 and IL-4 also play an important role in asthma.
  • Asthma is a common pulmonary inflammatory disease characterized by airway hyperresponsiveness (AHR), excessive mucus secretion, fibrosis and elevated IgE levels.
  • AHR airway hyperresponsiveness
  • Nonspecific stimuli such as cold air often lead to aggravated airway hyperresponsiveness.
  • AHR and excessive mucus secretion lead to airway obstruction, which is the main cause of death in asthma.
  • TH2 factors play an important role in the progression of asthma. Bronchial and alveolar lavage fluid of asthmatic patients overexpress IL-4 and IL-13.
  • IL-13 and IL-4 have certain functional similarities, some studies have shown that IL-13 plays a more important role in the progression of asthma than other Th2 cytokines.
  • IL-13 can promote goblet cell differentiation and fibrosis. Injection of recombinant IL-13 into the airways of mice that have not been stimulated by allergens will lead to airway inflammation, excessive mucus secretion and airway hyperresponsiveness. Injection of soluble IL13R ⁇ 2 can prevent the occurrence of AHR, excessive mucus secretion and lung inflammation in mice. Injection of IL-4R ⁇ antibody in asthma model can reduce AHR and eosinophils in bronchoalveolar lavage fluid. The study suggests that blocking IL-4R ⁇ may be effective in treating asthma.
  • Glucocorticoid is also the comparatively effective medicine for treating allergic diseases, inflammation etc.
  • known cortisol, corticosterone etc. are made in vivo glucocorticoid and dexamethasone, prednisone, prednisolone, budesonide etc.
  • glucocorticoid synthetic glucocorticoid.
  • These glucocorticoids are owing to having steroid structure, therefore are generally referred to as steroids, are applied in the treatment of various diseases.
  • steroids due to its use, sometimes show the side effects such as steroid peptic ulcer, steroid purpura, steroid pancreatitis, steroid diabetes, steroid cataract, steroid glaucoma etc.
  • ADCs Antibody drug conjugates
  • Most ADCs in preclinical and clinical development are used for oncology indications, where cytotoxic payloads target cancer cells expressing antigens.
  • modulation of pathogenic cell activity through ADC-mediated delivery of bioactive small molecules is also attractive for non-oncology indications, leading to the widespread application of this technology.
  • glucocorticoid drug conjugates such as WO2017210471, WO2019106609, WO2019136487, etc.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody drug conjugate and a counterion, wherein the antibody drug conjugate has a structure as shown in Formula I:
  • n 1 to 10.
  • the range of average drug load (n) can be the average number of glucocorticoid drugs bound to each 25G7-Fab, and non-limiting examples include the average number of glucocorticoid drugs bound to each 25G7-Fab being 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and any range between these point values.
  • it can be 2-8, 2-7, 2-6, 2-5, 2-4, 3-4, 3-5, 3.5-4.7, 5-6, 5-7, 5-8 and 6-8.
  • the average drug load (n) can be the mean of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.
  • n is a decimal or an integer.
  • the 25G7-Fab has a heavy chain as shown in SEQ ID NO: 11, and a light chain as shown in SEQ ID NO: 10.
  • the light chain variable region sequence of 25G7-Fab is shown in SEQ ID NO: 7, and the heavy chain variable region sequence is shown in SEQ ID NO: 8 shown.
  • hu25G7-A LCVR (hu25G7-A light chain variable region)
  • the light chain sequence of 25G7-Fab is shown in SEQ ID NO: 10, and the heavy chain sequence is shown in SEQ ID NO: 11; the heavy chain sequence of hu25G7 is shown in SEQ ID NO: 9, and the light chain sequence is shown in SEQ ID NO: 10.
  • the concentration of the antibody drug conjugate is 0.1 mg/mL-50 mg/mL in terms of protein concentration, non-limiting examples include 0.1 mg/mL, 0.5 mg/mL, 1.0 mg/mL, 1.5 mg/mL, 2.0 mg/mL, 2.5 mg/mL, 3.0 mg/mL, 3.5 mg/mL, 4.0 mg/mL, 4.5 mg/mL, 5.0 mg/mL, 6.0 mg/mL, 7.0 mg/mL, 8.0 mg/mL, 9.0 mg/mL, 10.0 mg/mL, 15.0 mg/mL, 20.0 mg/mL, 25.0 mg/mL, 30.0 mg/mL, 35.0 mg/mL, 40.0 mg/mL, 45.0 mg/mL, 50.0 mg/mL, and any range between these point values.
  • the concentration of the antibody drug conjugate is 0.5 mg/mL-10 mg/mL. In some embodiments, the concentration of the antibody drug conjugate is 1 mg/mL-5
  • the counterions include but are not limited to sulfate, phosphate, malate, maleate, oxalate, citrate, succinate, fumarate, itaconate, chloride, bromide, etc.
  • the counterions are selected from one or more of sulfate, citrate and chloride.
  • the counterions are selected from one or more of sodium citrate, sodium sulfate, zinc sulfate, magnesium sulfate, potassium sulfate, calcium sulfate, magnesium chloride, calcium chloride and zinc chloride.
  • the counterions are sodium sulfate and/or sodium citrate.
  • the concentration of the counterion is 0.01 mg/mL-10 mg/mL, non-limiting examples include 0.01 mg/mL, 0.05 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, 0.5 mg/mL, 0.6 mg/mL, 0.7 mg/mL, 0.8 mg/mL, 0.9 mg/mL, 1.0 mg/mL, 1.1 mg/mL, 1.2 mg/mL, 1.3 mg/mL, 1
  • the concentration of the counter ion is 0.05 mg/mL to 5 mg/mL. In some embodiments, the concentration of the counter ion is 0.1 mg/mL to 2 mg/mL.
  • the pharmaceutical composition further comprises a protective agent.
  • the protective agent is selected from amino acids and/or sugars.
  • the amino acid includes but is not limited to glycine, histidine, arginine, lysine, glutamic acid, alanine, valine, leucine, isoleucine, proline, tryptophan, phenylalanine, methionine, aspartic acid, etc.
  • the amino acid is glycine or histidine.
  • the concentration of the amino acid is 0.01 mg/mL-10 mg/mL, and non-limiting examples include 0.01 mg/mL, 0.05 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, 0.5 mg/mL, 0.6 mg/mL, 0.7 mg/mL, 0.8 mg/mL, 0.9 mg/mL, 1.0 mg/mL, 1.1 mg/mL, 1.2 mg/mL, 1.3 mg/mL, 1
  • the concentration of the amino acid is 0.05 mg/mL to 5 mg/mL. In some embodiments, the concentration of the amino acid is 0.1 mg/mL to 2 mg/mL.
  • the "sugar” of the present disclosure includes the conventional composition ( CH2O ) n and its derivatives, including monosaccharides, disaccharides, trisaccharides, polysaccharides, Sugar alcohol, reducing sugar, non-reducing sugar, etc.
  • the sugar includes but is not limited to sorbitol, mannitol, xylitol, trehalose, lactose, fructose, maltose, sucrose, etc.
  • the sugar is trehalose.
  • the concentration of the sugar is 0.01 mg/mL-10 mg/mL, non-limiting examples include 0.01 mg/mL, 0.05 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, 0.5 mg/mL, 0.6 mg/mL, 0.7 mg/mL, 0.8 mg/mL, 0.9 mg/mL, 1.0 mg/mL, 1.1 mg/mL, 1.2 mg/mL, 1.3 mg/mL, 1.
  • the sugar concentration is 0.05mg/mL-5mg/mL.
  • the sugar concentration is 0.1mg/mL-2mg/mL.
  • the pharmaceutical composition further comprises an organic solvent, including but not limited to methanol, ethanol, 1-propanol, isopropanol, tert-butanol, butanol, acetone, acetonitrile, acetic acid, ethyl acetate, methyl ethyl ketone, methyl tert-butyl ether, dimethyl sulfoxide, etc.
  • the organic solvent is isopropanol.
  • the amount of the organic solvent is 0.1%-50% v/v, and non-limiting examples include 0.1%, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 11.0%, 12.0%, 13.0%, 14.0%, 15.0%, 20.0%, 25.0%, 30.0%, 35.0%, 40.0%, 45.0%, 50.0%, and any range between these point values.
  • the amount of the organic solvent is 0.5%-30% v/v. In some embodiments, the amount of the organic solvent is 1%-10% v/v.
  • the pH of the pharmaceutical composition is 3.0-8.0, non-limiting examples include 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, and any range between these points.
  • the pH is 4.5-6.0.
  • the pH is 5.0 or 5.5.
  • the pharmaceutical composition comprises:
  • the pH of the pharmaceutical composition is 4.5-5.5.
  • the present disclosure also provides a lyophilized formulation containing an antibody drug conjugate, wherein the formulation can form the pharmaceutical composition as described above after reconstitution.
  • the present disclosure also provides a method for preparing a lyophilized preparation containing an antibody drug conjugate, comprising: The pharmaceutical composition is subjected to a freeze-drying step.
  • the present disclosure also provides a lyophilized preparation comprising an antibody drug conjugate, wherein the lyophilized preparation is obtained by freeze-drying the pharmaceutical composition of the antibody drug conjugate as described above.
  • the lyophilized preparation is in the form of microspheres.
  • the aerodynamic particle size of the microspheres is 0.01-10.0 ⁇ m. In some embodiments, the aerodynamic particle size of the microspheres is 0.5-5 ⁇ m.
  • the present disclosure also provides a reconstituted solution containing an antibody drug conjugate, wherein the reconstituted solution is prepared by reconstituted the lyophilized preparation as described above.
  • the present disclosure also provides a method for preparing the above-mentioned reconstituted solution, which comprises the step of reconstituted the above-mentioned lyophilized preparation, and the solution used for reconstitution is selected from but not limited to water for injection, physiological saline or glucose solution.
  • the present disclosure also provides a pharmaceutical composition, which comprises the aforementioned lyophilized preparation and a filler, wherein the filler includes but is not limited to trehalose, lactose, mannitol, glucose, sorbitol, dextran, etc.
  • the filler is trehalose.
  • the present disclosure also provides a product, which includes a container, wherein the pharmaceutical composition, lyophilized preparation, reconstituted solution or dry powder preparation as described above is contained in the container.
  • the container is a neutral borosilicate glass tube injection bottle.
  • the present disclosure also provides use of the aforementioned pharmaceutical composition, lyophilized preparation, reconstituted solution or product in the preparation of a medicament for treating or preventing an immune disease or disorder.
  • the present disclosure also provides a method for treating or preventing an immune disease or disorder, the method comprising administering an effective amount or a preventive effective amount of the aforementioned pharmaceutical composition, lyophilized preparation, reconstituted solution or preparation to a subject.
  • the disease or condition is selected from: asthma, nasal polyps, chronic sinusitis, allergic skin diseases, eosinophilic esophagitis, chronic obstructive pulmonary disease, allergic rhinitis, arthritis, inflammatory diseases, allergic reactions, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis and kidney disease.
  • the disease or condition is selected from asthma or allergic skin diseases.
  • Figure 1 Dose-response curve of whole-cell binding activity of the tested proteins to human IL-4R ⁇ expressing cells
  • FIG. 4 Time-concentration curves of ADC-2 and free Budesonide in various tissues after intratracheal administration
  • Figure 5 White blood cell and cell type count in bronchoalveolar lavage fluid of test case 6
  • Figure 6 White blood cell and cell type count in bronchoalveolar lavage fluid of test case 7
  • antibody-drug conjugate refers to a ligand connected to a biologically active drug through a stable linking unit.
  • ADC antibody-drug conjugate
  • ADC monoclonal antibody or antibody fragment connected to a biologically active glucocorticoid through a stable linking unit.
  • the antibody or antibody fragment can be combined with a glucocorticoid molecule containing a linker through a specific group therein (such as an interchain disulfide bond).
  • counterion refers to a charged or charge-polarized molecule that is capable of initiating microparticle formation from macromolecules such as proteins, nucleic acids, lipids, or oligosaccharides. Whether a charged molecule is a counterion can be empirically determined based on parameters including, but not limited to, the type of protein, pH, ionic strength, the type of organic solvent used, and the presence of salts and other ingredients such as active agents. As provided and described herein, a counterion can be anionic or a group with a net negative charge or charge polarization, cationic or a group with a net positive charge or charge polarization, or zwitterionic and a group with negative and positive charges or charge polarization.
  • microparticle is interchangeable with “microsphere” and refers to particles that include macromolecules and deliver an agent of interest, such as a drug or nutritional supplement to a subject, and whose size range (average length, width or diameter) is about or exactly 0.001 micrometer to about or exactly 500 micrometers.
  • the agent can be a macromolecule, such as a protein, nucleic acid, lipid or polysaccharide, or the macromolecule forming the microparticle can be a carrier for an active agent, such as a drug or nutritional supplement.
  • the microparticle can also include synthetic macromolecules, including polymers, such as polyethylene glycol (PEG), polylactic acid (PLA), polylactic-co-glycolic acid (PLGA) and natural polymers, such as albumin, gelatin, chitosan and dextran.
  • synthetic macromolecules including polymers, such as polyethylene glycol (PEG), polylactic acid (PLA), polylactic-co-glycolic acid (PLGA) and natural polymers, such as albumin, gelatin, chitosan and dextran.
  • the "microparticles" described herein can include and can be prepared from a specific natural or synthetic macromolecule, or from more than one type of the same natural or synthetic macromolecule (such as more than one type of protein), or from a combination of more than one different type of natural or synthetic macromolecule (such as a protein and a nucleic acid or a protein and a synthetic polymer).
  • microparticle as used herein also generally refers to a particle that is not a solid form of the entire solution from which it is produced, although frozen and/or dried particles of solutions containing macromolecules are also contemplated herein. Rather, a microparticle as used herein is generally a collection of a portion of the components of a solution, including salts, counterions, solvents and other ingredients, formed by processes including, but not limited to, precipitation, sedimentation, phase separation and colloid formation.
  • the monoclonal antibody molecular size variant determination method (CE-SDS) disclosed in the present invention can adopt the sodium dodecyl sulfate capillary electrophoresis (CE-SDS) ultraviolet detection method to quantitatively determine the purity of the recombinant monoclonal antibody product according to the capillary electrophoresis method (2015 edition of the "Chinese Pharmacopoeia" 0542) based on the molecular weight under reducing and non-reducing conditions.
  • 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 via a linker.
  • the number of drug molecules that can be coupled to the antibody in the coupling reaction will be less than the theoretical maximum value.
  • the loading capacity of the antibody-drug conjugate can be controlled by the following non-limiting methods, including:
  • amino acids that constitute 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, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, histidine, asparagine, glutamic acid, lysine, glutamine, methionine, arginine, serine, threonine, cysteine, proline, etc.
  • Non-natural amino acids such as citrulline.
  • non-natural amino acids do not constitute natural proteins and therefore do not participate in the synthesis of antibodies in the present disclosure.
  • the three letter codes and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).
  • the same class of Ig can be divided into different subclasses according to the difference in the amino acid composition of its hinge region and the number and position of the disulfide bonds of the heavy chain, such as IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • the light chain is divided into ⁇ chain or ⁇ chain according to the difference in the constant region.
  • Each of the five types of Ig can have ⁇ chain or ⁇ chain.
  • variable region The sequences of about 110 amino acids near the N-terminus of the antibody heavy and light chains vary greatly, which is the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable, which is the constant region.
  • the variable region includes three hypervariable regions (HVRs) and four relatively conservative framework regions (FRs). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining regions (CDRs).
  • CDRs complementarity determining regions
  • Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, arranged in the order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • murine antibody in this disclosure refers to antibodies prepared in mice according to the knowledge and skills in the art. When preparing, a test subject is injected with a specific antigen, and then a hybridoma expressing an antibody with the desired sequence or functional characteristics is isolated.
  • chimeric antibody refers to an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can reduce the immune response induced by the mouse antibody.
  • To establish a chimeric antibody it is necessary to first establish a hybridoma that secretes mouse-specific monoclonal antibodies, then clone the variable region gene from the mouse hybridoma cells, and then clone the constant region gene of the human antibody as needed, connect the mouse variable region gene with the human constant region gene into a chimeric gene, and then insert it into an expression vector, and finally express the chimeric antibody molecule in a eukaryotic system or a prokaryotic system.
  • humanized antibody also known as CDR-grafted antibody, refers to an antibody produced by transplanting mouse CDR sequences into human antibody variable region frameworks, that is, different types of human germline antibody framework sequences. It can overcome the heterologous response induced by chimeric antibodies due to carrying a large amount of mouse protein components.
  • framework sequences can be obtained from public DNA databases including germline antibody gene sequences or published references.
  • germline DNA sequences of human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk/vbase), and in Kabat, E.A. et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition.
  • human antibody variable region framework sequence can be subjected to minimal reverse mutations or back mutations to maintain activity.
  • Humanized antibodies of the present disclosure also include humanized antibodies further subjected to affinity maturation of CDR by phage display.
  • the literature further describing the method for humanization using mouse antibodies includes, for example, Queen et al., Proc., Natl. Acad. Sci. USA, 88, 2869, 1991 and the method of Winter and colleagues [Jones et al., Nature, 321, 522 (1986), Riechmann, et al., Nature, 332, 323-327 (1988), Verhoeyen, et al., Science, 239, 1534 (1988)].
  • Fully human antibody is antibodies whose variable and constant regions are both human, eliminating immunogenicity and toxic side effects.
  • the development of monoclonal antibodies has gone through four stages, namely: mouse 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 to an antigen. It has been shown that fragments of a full-length antibody can be used to perform the antigen-binding function of an 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, bivalent fragments comprising two Fab fragments connected by a disulfide bridge on the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VH and VL domains of a single arm of an antibody; (v) single domain or dAb fragments (Ward et al., (1989) Nature 341: 544-546), which consist of a VH domain; and (vi) isolated complementarity determining regions (CDRs) or (vii) combinations of two or more isolated CDRs, optionally connected by synthetic linkers.
  • CDRs complementarity determining regions
  • the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be connected by synthetic linkers using recombinant methods, so that they can be produced as a single protein chain in which the VL and VH regions are paired to form a monovalent molecule (called single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85: 5879-5883).
  • single-chain Fv single-chain Fv
  • Such single-chain antibodies are also intended to be included in the term "antigen-binding fragment" of an antibody.
  • Antigen-binding portions can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins.
  • the antibodies can be of different isotypes, for example, IgG (eg, IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibodies.
  • Fab is prepared by treating IgG antibody fragments with the protease papain (which cleaves amino acid residue 224 of the H chain). Among the fragments obtained, an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity is obtained, in which about half of the N-terminal side of the H chain and the entire L chain are bound together by a disulfide bond.
  • F(ab')2 is an antibody fragment having a molecular weight of about 100,000 and antigen-binding activity, obtained by digesting the portion below two disulfide bonds in the hinge region of IgG with the enzyme pepsin and comprising two Fab regions linked at the hinge position.
  • Fab' is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity obtained by cleaving the disulfide bond of the hinge region of the above-mentioned F(ab')2.
  • the Fab' fragment of the antibody can be produced by inserting a DNA encoding the Fab' fragment into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryotic or eukaryotic organism to express the Fab'.
  • single-chain antibody means a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker.
  • Such scFv molecules may have the general structure: NH2 - VL-linker-VH-COOH or NH2 - VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, for example variants using 1-4 repeats (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448).
  • 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.
  • the amino acid sequence boundaries of a CDR 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 (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3); the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3).
  • the CDR amino acid residues in VH are numbered 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) in human VL.
  • the CDR amino acid residues in VH are numbered approximately as 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3)
  • the CDR amino acid residues in VL are numbered approximately as 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 a 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 means being able to interact with human IL-4R (or its epitope, fragment).
  • the term "antigen binding site” herein refers to the three-dimensional site recognized by the antibody or antigen binding fragment herein.
  • binding refers to the binding of an antibody to a predetermined epitope on an antigen.
  • the antibody binds with an affinity (KD) of less than about 10-7 M, for example, less than about 10-8 M, 10-9 M or 10-10 M or less.
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells may include bacteria, microorganisms, plants or animal cells. Easily transformed bacteria include members of the family Enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae.
  • Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris.
  • Suitable animal host cell lines include CHO (Chinese Hamster Ovary Cell Line) and NS0 cells.
  • the engineered antibodies or antigen-binding fragments disclosed herein can be prepared and purified by 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 culture medium in a bioreactor to produce antibodies.
  • the culture fluid that secretes antibodies can be purified by conventional techniques. For example, purification is performed using an A or G Sepharose FF column containing an adjusted buffer.
  • Non-specifically bound components are washed away.
  • the bound antibodies are then eluted using a pH gradient method, and the antibody fragments are detected using SDS-PAGE and collected.
  • the antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange.
  • the resulting product must be immediately frozen, such as at -70°C, or freeze-dried.
  • anti-IL-4R antibody or "antibody that binds to IL-4R” refers to an antibody that is capable of binding to IL-4R, e.g., with sufficient affinity, such that the antibody can be used as a therapeutic agent targeting IL-4R.
  • the extent of binding of the anti-IL-4R antibody to unrelated, non-IL-4R proteins can be less than about 10% of the binding of the antibody to IL-4R as measured, e.g., by radioimmunoassay (RIA).
  • the antibody that binds to IL-4R has a dissociation constant (Kd) of ⁇ 1 ⁇ M, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, or ⁇ 0.1 nM.
  • peptide refers to a compound fragment between amino acids and proteins. It is composed of two or more amino acid molecules connected to each other by peptide bonds. It is a structural and functional fragment of protein. Hormones, enzymes, etc. are essentially peptides.
  • sucrose refers to a biological macromolecule composed of three elements: C, H, and O, which can be divided into monosaccharides, disaccharides, and polysaccharides.
  • “Pharmaceutical composition” means a mixture containing one or more antibody drug conjugates described herein or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients.
  • the purpose of the pharmaceutical composition is to maintain the stability of the antibody active ingredient, promote administration to the organism, and facilitate the absorption of the active ingredient to exert biological activity.
  • composition and “formulation” are not mutually exclusive.
  • compositions described in the present disclosure are in the form of solutions, and unless otherwise specified, the solvent therein is water.
  • “Lyophilized preparation” refers to a pharmaceutical composition in liquid or solution form or a preparation or pharmaceutical composition obtained after a liquid or solution preparation has been subjected to a vacuum freeze-drying step.
  • the terms “about” and “approximately” refer to values within an acceptable error range for a particular value determined by one of ordinary skill in the art, which depends in part on how it is measured or determined (i.e., the limitations of the measurement system). For example, “about” can mean within 1 or more than 1 standard deviation per practice in the art. Alternatively, “about” or “substantially comprising” can mean a range of up to 20%. Furthermore, particularly with respect to biological systems or processes, the term can mean up to one number. Unless otherwise stated, when specific values appear in the application and claims, the meaning of "about” or “substantially including” should be assumed to be within an acceptable error range for the specific value.
  • the numerical values in this disclosure are instrumental measurements or calculated values after instrumental measurements, and there is a certain degree of error. Generally speaking, plus or minus 10% is within the reasonable error range. Of course, the context in which the numerical value is used needs to be considered.
  • the total impurity content which is a value with an error change of no more than plus or minus 10% after measurement, can be plus or minus 9%, plus or minus 8%, plus or minus 7%, plus or minus 6%, plus or minus 5%, plus or minus 4%, plus or minus 3%, plus or minus 2% or plus or minus 1%, preferably plus or minus 5%.
  • Substituted means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms in the group are replaced independently of each other by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and the skilled person can determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, amino or hydroxy groups with free hydrogens may be unstable when combined with carbon atoms with unsaturated (e.g. olefinic) bonds.
  • administering and “treating” as applied to an animal, a human, a laboratory subject, a cell, a tissue, an organ, or a biological fluid, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, a human, a subject, a cell, a tissue, an organ, or a biological fluid.
  • administering and “treating” can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell.
  • administering and “treating” also mean in vitro and ex vivo treatment of, for example, a cell by an agent, a diagnostic, a combination composition, or by another cell.
  • Treatment as applied to humans, veterinary medicine, or research subjects refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent, such as a composition comprising any of the binding compounds of the present disclosure, to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect.
  • the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the treated patient or population to induce regression of such symptoms or inhibit the development of such symptoms to any clinically measurable degree.
  • the amount of therapeutic agent effective to alleviate any specific disease symptom may vary according to a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient.
  • Whether the disease symptom has been alleviated can be evaluated by any clinical test method commonly used by physicians or other professional health care personnel to evaluate the severity or progression of the symptom.
  • the embodiments of the present disclosure e.g., treatment methods or products
  • may not be effective in alleviating every target disease symptom they should alleviate the target disease symptoms in a statistically significant number of patients as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.
  • an "effective amount” includes an amount sufficient to ameliorate or prevent symptoms or conditions of a medical disease.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a particular patient or veterinary subject may vary depending on factors such as the type of The condition being treated, the patient's overall health, the method and dosage of administration, and the severity of side effects.
  • An effective amount may be the maximum dose or administration regimen that avoids significant side effects or toxic effects.
  • Dislacement refers to the replacement of the solvent system for dissolving the antibody protein or antibody drug conjugate, for example, using the buffer system of the stable preparation to replace the high salt or hypertonic solvent system containing the antibody protein or antibody drug conjugate by physical manipulation, so that the antibody protein or antibody drug conjugate is present in the stable preparation.
  • the so-called physical manipulation method includes but is not limited to ultrafiltration, dialysis or centrifugation followed by re-dissolution.
  • NMR shift ( ⁇ ) is given in units of 10 -6 (ppm). NMR measurements were performed using a Bruker AVANCE-400 NMR spectrometer, with deuterated dimethyl sulfoxide (DMSO-d 6 ), deuterated chloroform (CDCl 3 ), deuterated methanol (CD 3 OD) as the measurement solvent, and tetramethylsilane (TMS) as the internal standard.
  • DMSO-d 6 deuterated dimethyl sulfoxide
  • CDCl 3 deuterated chloroform
  • CD 3 OD deuterated methanol
  • TMS tetramethylsilane
  • MS was measured using Shimadzu 2010 Mass Spectrometer or Agilent 6110A MSD mass spectrometer.
  • HPLC determination was performed using Shimadzu LC-20A systems, Shimadzu LC-2010HT series or Agilent 1200 LC high pressure liquid chromatograph (Ultimate XB-C18 3.0*150mm column or Xtimate C18 2.1*30mm column1.
  • Chiral HPLC analysis was performed using 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., 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 columns; thin layer chromatography silica gel plates use Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates, the silica gel plates used in thin layer chromatography (TLC) use specifications of 0.15mm ⁇ 0.2mm, and the specifications used for thin layer chromatography
  • the chiral preparative column used was DAICEL CHIRALPAK IC (250mm*30mm, 10um) or Phenomenex-Amylose-1 (250mm*30mm, 5um).
  • the CombiFlash rapid preparation instrument uses Combiflash Rf150 (TELEDYNE ISCO).
  • the average kinase inhibition rate and IC50 value were determined using NovoStar microplate reader (BMG, Germany).
  • the known starting materials disclosed herein can be synthesized by methods known in the art, or can be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc, Darui Chemicals, and other companies.
  • the reactions can 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 capacity of about 1L.
  • Hydrogen atmosphere means that the reaction bottle is connected to a hydrogen balloon with a capacity of about 1L.
  • the pressurized hydrogenation reaction uses a Parr 3916EKX hydrogenator and a Clearland QL-500 hydrogen generator or a HC2-SS hydrogenator.
  • the hydrogenation reaction is usually carried out by evacuating the vacuum, filling with hydrogen, and repeating the operation three times.
  • Microwave reactions were performed using a CEM Discover-S 908860 microwave reactor.
  • the solution refers to an aqueous solution.
  • reaction temperature is room temperature, 20°C to 30°C.
  • the reaction progress in the embodiment is monitored by thin layer chromatography (TLC), the developing solvent used in the reaction, the eluent system of column chromatography used for purifying the compound and the developing solvent system of thin layer chromatography include: 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 adjusted according to the polarity of the compound, and a small amount of alkaline or acidic reagents such as triethylamine and acetic acid can also be added for adjustment.
  • TLC thin layer chromatography
  • DAST diethylaminosulfur trifluoride
  • THF tetrahydrofuran
  • NMP N-methylpyrrolidone
  • DCM dichloromethane
  • m-CPBA m-chloroperbenzoic acid
  • DIEA N,N-diisopropylethylamine
  • TEA triethylamine
  • Boc tert-butyloxycarbonyl, MeOH: methanol
  • Et 2 O diethyl ether.
  • Example 1 Preparation of N-((10S)-10-benzyl-1-((6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-7-hydroxy-6a, 8a-dimethyl-4-oxo-10-propyl-1,2,4,6a, 6b, 7,8,8a, 11a, 12,12a, 12b-dodecahydro-8bH-naphtho[2′, 1′: 4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-1,6,9,12,15-pentaoxo-3-oxo-5,8,11,14-tetraethylene glycol-16-yl)-1-(2-bromoacetylamino)-3,6,9,12-tetraoxopentadecane-15-amide (Compound 1)
  • Step 5 Preparation of (9H-fluoren-9-yl)methyl (2-(((2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxolan-8b-yl)-2-oxoethoxy)methyl)amino)-2-oxoethyl)carbamate (Compound 1f)
  • Step 6) Preparation of 2-amino-N-((2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxolan-8b-yl)-2-oxoethoxy)methyl)acetamide (Compound 1g)
  • Step 7 Preparation of (9H-fluoren-9-yl)methyl ((10S)-10-benzyl-1-((6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-7-hydroxy-6a, 8a-dimethyl-4-oxo-10-propyl-1,2,4,6a, 6b, 7,8,8a, 11a, 12, 12a, 12b-dodecahydro-8bH-naphtho[2′, 1′: 4,5]indeno[1,2-d][1,3]dioxolan-8b-yl)-1,6,9,12,15,18-hexaoxo-3,21,24,27,30-pentaoxo-5,8,11,14,17-pentahydrotriacontan-32-yl)carbamate (Compound 1h)
  • Step 8) Preparation of 1-amino-N-((10S)-10-benzyl-1-((6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-7-hydroxy-6a, 8a-dimethyl-4-oxo-10-propyl-1,2,4,6a, 6b, 7,8,8a, 11a, 12,12a, 12b-dodecahydro-8bH-naphtho[2′, 1′: 4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-1,6,9,12,15-pentaoxo-3-oxo-5,8,11,14-polytetraethylene glycol-16-yl-3,6,9,12-tetraoxopentadecane-15-amide (Compound 1i)
  • Step 9) Preparation of N-((10S)-10-benzyl-1-((6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-7-hydroxy-6a, 8a-dimethyl-4-oxo-10-propyl-1,2,4,6a, 6b, 7,8,8a, 11a, 12,12a, 12b-dodecahydro-8bH-naphtho[2′, 1′: 4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-1,6,9,12,15-pentaoxo-3-oxo-5,8,11,14-tetraethylene glycol-16-yl)-1-(2-bromoacetylamino)-3,6,9,12-tetraoxopentadecane-15-amide (Compound 1)
  • Embodiment 2 is a diagrammatic representation of Embodiment 1:
  • Step 2 Preparation of (9H-fluoren-9-yl)methyl ((10S)-10-benzyl 1-((6aR, 6bS, 7S, 8aS, 8bS, 11aR, 12aS, 12bS)-7-hydroxy-6a, 8a-dimethyl-4-oxo-10-propyl-1,2,4,6a, 6b, 7,8,8a, 11a, 12,12a, 12b-dodecahydro-8bH-naphtho[2′, 1′: 4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-1,6,9,12,15-pentaoxo-3-oxo-5,8,11,14-tetraethylene 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 for expansion culture.
  • the clones were inoculated in the culture medium for expansion culture, and the plasmid containing the deoxyribonucleic acid sequence of the 25G7-Fab antibody was extracted in large quantities.
  • liposome transfection the expression plasmid was mixed with the transfection reagent and added to Expi 293 cells.
  • the cell culture was harvested after 5 days of culture in a constant temperature incubator. The cell culture was centrifuged and the supernatant was taken, the cell debris was filtered to remove the clear liquid.
  • the target protein was captured using a kappa select chromatography column.
  • the target protein was filtered, and the A280 absorbance value was measured by Nanodrop and divided by the extinction coefficient to obtain the protein concentration, which was multiplied by the volume to determine the final yield.
  • the protein was subjected to SDS-PAGE, SEC-HPLC and endotoxin detection by gel electrophoresis and size exclusion chromatography, and the final product was 25G7-Fab.
  • AZD1402-TAG the protein sequence comes from SEQ ID NO1 in patent WO2020200960A1. In order to facilitate protein preparation and purification, a His tag was fused to the C-terminus of the sequence and was finally named AZD1402-TAG).
  • the coding gene sequence of the above protein was synthesized and subcloned into pcDNA3.4.
  • the expression plasmid was mixed with the transfection reagent and incubated at 37°C for 15 minutes. The mixture was added dropwise to the HEK293 cell solution. After the cell solution was cultured on a shaker at 37°C for one week, the cell culture was centrifuged and the supernatant was taken.
  • the nickel affinity chromatography column was equilibrated with an imidazole-free buffer, and then the protein sample was passed through the nickel affinity chromatography column for loading. After equilibration with an imidazole-free buffer again, the column-bound protein was eluted with a buffer containing a high concentration of imidazole. The eluted protein was transferred to a dialysis bag and 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 KH2PO4 ( 8.50g ), K2HPO4 (8.56g), NaCl (5.86g) and EDTA (1.50g), add 1.6L water for injection, stir for half an hour, and then dilute to 2.0L with water for injection after complete dissolution. The pH is measured to be 6.30 ⁇ 0.1.
  • TCEP tris(2-carboxyethyl)phosphine
  • TCEP tris(2-carboxyethyl)phosphine
  • Test Example 1 Affinity detection of antibody drug conjugate ADC-2 and human IL-4R ⁇ protein
  • SPR Surface plasmon resonance
  • the flow rate was set to 30 ⁇ L/min.
  • the detection time of ADC-2, 25G7-Fab and AZD1402-TAG was 100 seconds for binding and 600 seconds for dissociation.
  • Biacore 8K evaluation software (Cytiva) was used for analysis to obtain the affinity data of each test protein.
  • the results are shown in Table 1.
  • the KD value of ADC-2 binding to human IL-4R ⁇ antigen protein is 11.0 pM, which is slightly better than AZD1402-TAG and 25G7 - Fab.
  • Test Example 2 Whole-cell binding activity of antibody-drug conjugate ADC-2 with human IL-4R ⁇ expressing cells
  • Antibody drug conjugate ADC-2, 25G7-Fab, hu25G7 (the heavy chain sequence is shown in SEQ ID NO: 9, the light chain sequence is shown in SEQ ID NO: 10), AZD1402-TAG.
  • TF-1 and Karpas 299 cell lines were identified to express IL-4R ⁇ . After collecting TF-1 and Karpas299 cells in the logarithmic growth phase, the cell density was adjusted and plated at 1 ⁇ 10 5 cells/well in a round-bottom 96-well plate. The cells were incubated with different concentrations of anti-IL-4R antibodies (25G7, naked anti-25G7-Fab), antibody-drug conjugate ADC-2 and AZD1402-TAG at 4°C for 45 minutes, and the antibodies were washed away with FACS Buffer (PBS containing 2% FBS) and then added. Fluorescently labeled secondary antibodies were used for staining.
  • FACS Buffer PBS containing 2% FBS
  • 25G7-Fab, hu25G7 and ADC-2 were stained with FITC-conjugated anti-human IgG (Fab specific) secondary antibodies (Sigma, CAT#F5512-1ML), and AZD1402-TAG was stained with FITC-conjugated anti-His antibody (GenScript, CAT#A01620).
  • the cells were incubated with the secondary antibodies at 4°C for 30 minutes, then washed twice with FACS Buffer, and detected by flow cytometry (BD, FACS Celesta). The detection results were analyzed using FlowJo (FlowJo, LLC) software.
  • Table 2 EC50 values of whole cell binding activity of the tested proteins to human IL-4R ⁇ expressing cells
  • the results are shown in Figure 1 and Table 2.
  • the EC 50 value of ADC-2 binding to TF-1 cells is 0.107 nM, which is comparable to naked antibody 25G7-Fab and hu25G7.
  • AZD1402-TAG has a strong binding effect on TF-1 cells, but due to different detection antibodies, the data are not comparable with ADC-2, 25G7-Fab, etc.
  • Test Example 3 Blocking effect of antibody drug conjugate ADC-2 on IL-4/IL-13 signaling pathway
  • Activation of STAT6 is a key step in the activation of the IL-4/IL-13 signaling pathway.
  • the blocking effect of ADC-2 on the 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-i1413), in which the human STAT6 gene and the secreted alkaline phosphatase reporter gene (Secreted alkaline phos-phatase, SEAP) induced by phosphorylated STAT6 were overexpressed.
  • the SEAP substrate QUANTI-Blue can be used to detect the content of SEAP secreted in the cell culture supernatant to evaluate the activation level of the IL-4/IL-13 signaling pathway.
  • the density was adjusted to 5 ⁇ 10 5 cells/ml, and 100 ⁇ L was added to each well of a 96-well flat-bottom culture plate, and cultured at 37°C, 5% CO 2 for 24 hours. Then, 20 ⁇ L of the gradient diluted test protein and 20 ⁇ L of recombinant human IL-4 or IL-13 were added to each well, and the cell culture plate was placed in a 37°C, 5% CO 2 incubator for further incubation for 20 to 24 hours.
  • Test Example 4 Endocytosis activity of antibody drug conjugate ADC-2 in TF-1 cells
  • TF-1 cells with good growth status were collected and adjusted to a density of 1 ⁇ 10 6 cells/mL
  • 100 ⁇ L/well was added to a 96-well cell culture plate and placed at 4°C.
  • Each test protein was added at a final concentration of 2 nM.
  • a single culture plate was set at each time point. During the period, the culture plate was taken out according to different incubation times, washed with FACS buffer and centrifuged, and then fluorescently labeled secondary antibodies were added for incubation.
  • 25G7-Fab, hu25G7 and ADC-2 were labeled with FITC-conjugated anti-human IgG (Fab-specific) secondary antibodies, and AZD1402-TAG was labeled with FITC-conjugated anti-His antibodies.
  • the cells were incubated with the secondary antibody at 4°C for 30 minutes, then washed twice with FACS Buffer, and detected by flow cytometry (BD, FACS Celesta). The detection results were analyzed using FlowJo (FlowJo, LLC) software.
  • the formula for calculating the endocytosis rate at a specific time point is:
  • Test Example 5 Pharmacokinetic test of antibody drug conjugate ADC-2 in mouse model
  • mice received intratracheal aerosol administration (it), and 300-400 ⁇ L of blood was collected from the jaw at different time points after administration. After standing for 2 hours, the blood was centrifuged at 7500 rpm and 4°C for 10 minutes to collect serum. After being drunk and fixed, the neck muscles of the mice were bluntly separated to expose the trachea. The trachea was opened laterally, and the lavage needle was slowly inserted. The pre-drawn 4°C pre-cooled saline was slowly pushed into the lung tissue for lavage, and the alveolar lavage fluid (BALF) was collected. A total of 3 lavages were performed, with 300 ⁇ L of saline used for the first two lavages and 400 ⁇ L used for the third lavage.
  • BALF alveolar lavage fluid
  • the collected alveolar lavage fluid was centrifuged at 1000 rpm and 4°C for 10 minutes, and the supernatant was collected. After euthanizing the mice, the mouse chest cavity was opened, the complete mouse lung tissue was separated and collected, the blood stains on the surface of the lung tissue were washed off with PBS, and the lung tissue was weighed after the surface PBS was dried with a paper towel.
  • the lung tissue was placed in a 2 mL centrifuge tube, 1 mL of cell lysis buffer (CST, #9803) was added, and the homogenizer (Shanghai Jing Xin, Tissuelyser-48) was used to grind for 2 min at 60 Hz, and then centrifuged at 10,000 ⁇ g at 4 ° C for 10 min. The supernatant was the lung tissue homogenate, and the protein concentration of the homogenate was detected (Pierce, 23227). Serum, BALF and lung tissue homogenate were stored at -80 ° C. The dosing regimen is shown in Table 4, and the sampling regimen is shown in Table 5.
  • Sample incubation Add 100 ⁇ L of diluted serum, BALF or lung tissue homogenate to the ELISA plate and incubate at room temperature at 400 rpm for 1.5 h.
  • Detection Dilute the detection antibody (Invitrogen, A18811) 10,000-fold with assay buffer (0.05% PBST containing 0.5% BSA) and add to the ELISA plate, 100 ⁇ L per well, and incubate at room temperature at 400 rpm for 1 h.
  • Termination Add 100 ⁇ L of substrate reaction termination solution (Solarbio, C1058) to each well and detect the light absorbance at a wavelength of 620 nm.
  • Test Example 6 In vivo efficacy of ADC-2 in OVA-induced mouse asthma model
  • mice were randomly divided into normal control animals and modeling animals.
  • 100 ⁇ L of PBS solution containing 200 ⁇ g OVA (Sigma, A5503) was intraperitoneally injected, and an equal volume of aluminum hydroxide was used as an adjuvant to sensitize the mice.
  • the mice On Day 21-27, the mice inhaled a 3% OVA aerosol solution once a day for 35 minutes each time. Starting on Day 21, half an hour before inhaling the OVA aerosol solution, the mice received intratracheal aerosol administration.
  • the specific dosing regimen is shown in Table 7.
  • mice On Day 28, after the mice were euthanized, the neck muscles of the mice were bluntly separated to expose the trachea. The trachea was opened laterally, and the lavage needle was slowly inserted. The pre-drawn 4°C pre-cooled saline was slowly pushed into the lung tissue for lavage, and the alveolar lavage fluid was collected. A total of 3 lavages were performed, with 300 ⁇ L of saline used for the first two lavages and 400 ⁇ L used for the third lavage. The cells were centrifuged at 1,000 rpm and 4°C for 10 min. The cell pellet was resuspended with 350 ⁇ L of pre-cooled saline and then centrifuged with an automatic blood flow analyzer. The white blood cell and cell type counts were performed using a blood analyzer.
  • the experimental results are shown in Figure 5.
  • the antibody-drug conjugate ADC-2 significantly reduced the number of leukocytes, eosinophils and neutrophils in BALF, and also inhibited the number of monocytes to a certain extent.
  • the efficacy was better than AZD1402-TAG, and significantly better than 25G7-Fab and Budesonide.
  • ADC-2 showed the synergistic effect of 25G7-Fab and Budesonide in terms of efficacy.
  • Test Example 7 Dose exploration of the in vivo efficacy of ADC-2 in the OVA-induced mouse asthma model
  • Antibody drug conjugate ADC-2, AZD1402-TAG, Budesonide is provided.
  • 10 ⁇ g ADC-2 can significantly reduce the number of eosinophils in the alveolar lavage fluid, which is equivalent to AZD1402-TAG and slightly better than budesonide, which is far higher than the clinical equivalent dose.
  • 10 ⁇ g ADC-2 also has a very significant ability to inhibit the number of white blood cells, and also has a certain degree of inhibition on the number of monocytes and neutrophils.
  • the particle size was measured using a SYMPATEC particle size analyzer with a dispersion pressure of 4 bar. The particle size results are shown in Table 10.
  • the prepared drug-containing inhalation dry powder was filled into transparent HPMC capsules (No. 3) with a loading volume of 10 mg.
  • the capsule samples were subjected to APSD testing using the NGI impactor of COPLEY Company in accordance with the provisions of the General Rules 0951 [Aerodynamic Properties of Fine Particles of Inhalation Preparations] of the 2015 edition of the Chinese Pharmacopoeia.
  • the APSD distribution results at all levels were input into the CITDAS version 3.10 software (COPLEY) to obtain the FPF ( ⁇ 5 ⁇ m), MMAD, GSD and other values. The results are shown in Table 11.
  • Example 8 Preparation of inhalation dry powders e, f, and g
  • the particle size was measured using a SYMPATEC particle size analyzer with a dispersion pressure of 4 bar. The particle size results are shown in Table 14.
  • Example 7 The same preparation and freeze-drying process as in Example 7 was used to prepare inhalation powder according to the prescription composition in Table 15.
  • the particle size was measured using a SYMPATEC particle size analyzer with a dispersion pressure of 4 bar.
  • the particle size results are shown in Table 16. The results showed that the geometric particle size did not change much when the drug concentration increased from 2 mg/ml to 3.5 mg/ml.
  • the aerodynamic particle size determination method was the same as in Example 7, and the APSD results are shown in Table 17. The results showed that the drug concentration within the range of 2 to 3.5 mg/ml had little effect on the aerodynamic particle size results such as FPF and MMAD.
  • inhalation dry powders c and f were both composed of several nano-scale smooth spheres.
  • Preparation of inhalation dry powder m (0.4 mg): weigh the amount of histidine, sodium citrate and 0.06 mg trehalose, add water to prepare an auxiliary material solution, add the prescribed amount of ADC-2 buffer (prepared in Example 6), gently stir evenly, add the prescribed amount of isopropanol, and gently stir evenly.
  • the aerodynamic particle size determination method is the same as in Example 7, and the APSD results are shown in Table 19.

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Abstract

一种含有糖皮质激素的抗体药物偶联物的药物组合物。具体涉及一种药物组合物，包含抗体药物偶联物和抗衡离子。药物组合物具备良好的空气动力学特征，适合吸入给药。

Description

一种含有糖皮质激素的抗体药物偶联物的药物组合物
本申请要求申请日为2023/7/28的中国专利申请2023109382848的优先权。本申请引用上述中国专利申请的全文。
技术领域
本公开属于药物制剂领域，具体涉及一种含有糖皮质激素的抗体药物偶联物的药物组合物。
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
本公开提供一种药物组合物，包含抗体药物偶联物和抗衡离子，其中所述抗体药物偶联物具有如式I所示的结构：
其中：
25G7-Fab包含重链可变区和轻链可变区，所述重链可变区包含：如SEQ ID NO：1或SEQ ID NO：12所示的HCDR1，如SEQ ID NO：2所示的HCDR2，如SEQ ID NO：3所示的HCDR3，所述轻链可变区包含：如SEQ ID NO：4所示的LCDR1，如SEQ ID NO：5所示的LCDR2，如SEQ ID NO：6所示的LCDR3；
n为1至10。
在一些实施方案中，平均药物载量(n)的范围可以是每个25G7-Fab结合糖皮质激素药物的平均数，非限制性的实施例包括每个25G7-Fab结合糖皮质激素药物的平均个数为1、2、3、4、5、6、7、8、9、10以及这些点值之间的任意范围。例如可以是2-8，2-7，2-6，2-5，2-4，3-4，3-5，3.5～4.7，5-6，5-7，5-8和6-8。示例性的，平均药物载量(n)可以为1，2，3，4，5，6，7，8，9，10的均值。n是小数或整数。
在一些实施方案中，所述25G7-Fab具有如SEQ ID NO：8所示的重链可变区，和如SEQ ID NO：7所示的轻链可变区。
在一些实施方案中，所述25G7-Fab具有如SEQ ID NO：11所示的重链，和如SEQ ID NO：10所示的轻链。
25G7-Fab的CDR序列见表1：
表1 25G7-Fab的CDR序列
25G7-Fab的轻链可变区序列如SEQ ID NO：7所示，重链可变区序列如SEQ ID NO：8 所示。
hu25G7-A LCVR(hu25G7-A轻链可变区)
SEQ ID NO：7
hu25G7-VH(hu25G7重链可变区)
SEQ ID NO：8
25G7-Fab的轻链序列如SEQ ID NO：10所示，重链序列如SEQ ID NO：11所示；hu25G7的重链序列如SEQ ID NO：9所示，轻链序列如SEQ ID NO：10所示。
hu25G7-IgG4 HC
SEQ ID NO：9
hu25G7-A LC
SEQ ID NO：10
hu25G7-24-IgG4 VH-CH1
SEQ ID NO：11
在一些实施方案中，所述抗体药物偶联物的浓度以蛋白浓度计为0.1mg/mL-50mg/mL，非限制性的实施例包括0.1mg/mL、0.5mg/mL、1.0mg/mL、1.5mg/mL、2.0mg/mL、2.5mg/mL、3.0mg/mL、3.5mg/mL、4.0mg/mL、4.5mg/mL、5.0mg/mL、6.0mg/mL、7.0mg/mL、8.0mg/mL、9.0mg/mL、10.0mg/mL、15.0mg/mL、20.0mg/mL、25.0mg/mL、30.0mg/mL、35.0mg/mL、40.0mg/mL、45.0mg/mL、50.0mg/mL，以及这些点值之间的任意范围。在一些实施方案中，抗体药物偶联物的浓度为0.5mg/mL-10mg/mL。在一些实施方案中，抗体药物偶联物的浓度为1mg/mL-5mg/mL。
在一些实施方案中，所述抗衡离子包括但不限于硫酸盐、磷酸盐、苹果酸盐、马来酸盐、草酸盐、柠檬酸盐、琥珀酸盐、富马酸盐、衣康酸盐、氯化物、溴化物等。在一些实施方案中，所述抗衡离子选自硫酸盐、柠檬酸盐和氯化物中的一种或多种。在一些实施方案中，所述抗衡离子选自柠檬酸钠、硫酸钠、硫酸锌、硫酸镁、硫酸钾、硫酸钙、氯化镁、氯化钙和氯化锌中的一种或多种。在一些实施方案中，所述抗衡离子为硫酸钠和/或柠檬酸钠。
在一些实施方案中，所述抗衡离子的浓度为0.01mg/mL-10mg/mL，非限制性的实施例包括0.01mg/mL、0.05mg/mL、0.1mg/mL、0.2mg/mL、0.3mg/mL、0.4mg/mL、0.5mg/mL、0.6mg/mL、0.7mg/mL、0.8mg/mL、0.9mg/mL、1.0mg/mL、1.1mg/mL、1.2mg/mL、1.3mg/mL、1.4mg/mL、1.5mg/mL、1.6mg/mL、1.7mg/mL、1.8mg/mL、1.9mg/mL、2.0mg/mL、2.2mg/mL、2.4mg/mL、2.6mg/mL、2.8mg/mL、3.0mg/mL、4.0mg/mL、5.0mg/mL、6.0mg/mL、7.0mg/mL、8.0mg/mL、9.0mg/mL、10.0mg/mL，以及这些点值之间的任意范围。在一些实施方案中，所述抗衡离子的浓度为0.05mg/mL-5mg/mL。在一些实施方案中，所述抗衡离子的浓度为0.1mg/mL-2mg/mL。
在一些实施方案中，所述药物组合物还包含保护剂。在一些实施方案中，所述保护剂选自氨基酸和/或糖。
在一些实施方案中，所述氨基酸包括但不限于甘氨酸、组氨酸、精氨酸、赖氨酸、谷氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、色氨酸、苯丙氨酸、甲硫氨酸、天门冬氨酸等。在一些实施方案中，所述氨基酸为甘氨酸或组氨酸。
在一些实施方案中，所述氨基酸的浓度为0.01mg/mL-10mg/mL，非限制性的实施例包括0.01mg/mL、0.05mg/mL、0.1mg/mL、0.2mg/mL、0.3mg/mL、0.4mg/mL、0.5mg/mL、0.6mg/mL、0.7mg/mL、0.8mg/mL、0.9mg/mL、1.0mg/mL、1.1mg/mL、1.2mg/mL、1.3mg/mL、1.4mg/mL、1.5mg/mL、1.6mg/mL、1.7mg/mL、1.8mg/mL、1.9mg/mL、2.0mg/mL、2.2mg/mL、2.4mg/mL、2.6mg/mL、2.8mg/mL、3.0mg/mL、4.0mg/mL、5.0mg/mL、6.0mg/mL、7.0mg/mL、8.0mg/mL、9.0mg/mL、10.0mg/mL，以及这些点值之间的任意范围。在一些实施方案中，所述氨基酸的浓度为0.05mg/mL-5mg/mL。在一些实施方案中，所述氨基酸的浓度为0.1mg/mL-2mg/mL。
本公开的“糖”包含常规组合物(CH2O)n及其衍生物，包括单糖，二糖，三糖，多糖， 糖醇，还原性糖，非还原性糖等等。在一些实施方案中，所述的糖包括但不限于山梨醇、甘露醇、木糖醇、海藻糖、乳糖、果糖、麦芽糖、蔗糖等。在一些实施方案中，所述的糖为海藻糖。
在一些实施方案中，所述糖的浓度为0.01mg/mL-10mg/mL，非限制性的实施例包括0.01mg/mL、0.05mg/mL、0.1mg/mL、0.2mg/mL、0.3mg/mL、0.4mg/mL、0.5mg/mL、0.6mg/mL、0.7mg/mL、0.8mg/mL、0.9mg/mL、1.0mg/mL、1.1mg/mL、1.2mg/mL、1.3mg/mL、1.4mg/mL、1.5mg/mL、1.6mg/mL、1.7mg/mL、1.8mg/mL、1.9mg/mL、2.0mg/mL、2.2mg/mL、2.4mg/mL、2.6mg/mL、2.8mg/mL、3.0mg/mL、4.0mg/mL、5.0mg/mL、6.0mg/mL、7.0mg/mL、8.0mg/mL、9.0mg/mL、10.0mg/mL，以及这些点值之间的任意范围。在一些实施方案中，所述糖的浓度为0.05mg/mL-5mg/mL。在一些实施方案中，所述糖的浓度为0.1mg/mL-2mg/mL。
在一些实施方案中，所述药物组合物还包含有机溶剂，所述有机溶剂包括但不限于甲醇、乙醇、1-丙醇、异丙醇、叔丁醇、丁醇、丙酮、乙腈、乙酸、乙酸乙酯、甲基乙基酮、甲基叔丁基醚、二甲基亚砜等。在一些实施方案中，所述有机溶剂为异丙醇。
在一些实施方案中，所述有机溶剂的量为0.1％-50％v/v，非限制性的实施例包括0.1％、0.5％、1.0％、1.5％、2.0％、2.5％、3.0％、3.5％、4.0％、4.5％、5.0％、5.5％、6.0％、6.5％、7.0％、7.5％、8.0％、8.5％、9.0％、9.5％、10.0％、11.0％、12.0％、13.0％、14.0％、15.0％、20.0％、25.0％、30.0％、35.0％、40.0％、45.0％、50.0％，以及这些点值之间的任意范围。在一些实施方案中，所述有机溶剂的量为0.5％-30％v/v。在一些实施方案中，所述有机溶剂的量为1％-10％v/v。
在一些实施方案中，所述药物组合物的pH为3.0-8.0，非限制性的实施例包括3.5、3.6、3.7、3.8、3.9、4.0、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.8、4.9、5.0、5.1、5.2、5.3、5.4、5.5、5.6、5.7、5.8、5.9、6.0、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9、7.0、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9、8.0，以及这些点值之间的任意范围。在一些实施方案中，pH为4.5-6.0。在一些实施方案中，pH为5.0或5.5。
在一些实施方案中，所述的药物组合物，包含：
a)以蛋白浓度计，1mg/mL-5mg/mL的所述抗体药物偶联物，
b)0.1mg/mL-2mg/mL的抗衡离子，所述抗衡离子为柠檬酸钠或硫酸钠，
c)0.1mg/mL-2mg/mL的氨基酸，所述氨基酸为组氨酸或甘氨酸，
d)0.1mg/mL-2mg/mL的糖，所述糖为海藻糖，
e)1％-10％v/v的有机溶剂，所述有机溶剂为异丙醇；
所述药物组合物的pH为4.5-5.5。
本公开还提供一种含抗体药物偶联物的冻干制剂，其中所述制剂复溶后可形成如上所述的药物组合物。
本公开还提供一种制备含抗体药物偶联物的冻干制剂的方法，其中包括将如上所述的 药物组合物经冷冻干燥的步骤。
本公开还提供一种包含抗体药物偶联物的冻干制剂，所述冻干制剂通过将如上所述的抗体药物偶联物的药物组合物冷冻干燥获得。
在一些实施方案中，所述冻干制剂的形态为微球。在一些实施方案中，所述微球的空气动力学粒径为0.01-10.0μm。在一些实施方案中，所述微球的空气动力学粒径为0.5-5μm。
本公开还提供一种含抗体药物偶联物的复溶溶液，其中所述复溶溶液是通过将如上所述的冻干制剂复溶制备获得。
本公开还提供制备上述复溶溶液的方法，其中包括将前述冻干制剂经复溶的步骤，其复溶所用溶液选自但不限于注射用水、生理盐水或葡萄糖溶液。
本公开还提供了一种药物组合物，其包含前述冻干制剂，以及填充剂，所述填充剂包括但不限于海藻糖、乳糖、甘露醇、葡萄糖、山梨醇、右旋糖酐等。在一些实施方案中，所述填充剂为海藻糖。
本公开还提供一种制品，其包括容器，该容器中装有如上所述的药物组合物、冻干制剂、复溶溶液或干粉制剂。在一些实施方案中，该容器为中性硼硅玻璃管制注射剂瓶。
本公开还提供前述的药物组合物、冻干制剂、复溶溶液或制品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。
本公开还提供一阵治疗或预防免疫性疾病或病症的方法，所述方法包括向受试者施用有效量或预防有效量的前述药物组合物、冻干制剂、复溶溶液或制品。
在一些实施方案中，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。在一些实施方案中，所述疾病或病症选自哮喘或过敏性皮肤病。
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
图7：吸入干粉处方c的扫描电镜图
图8：吸入干粉处方f的扫描电镜图
术语
为了更容易理解本公开，以下具体定义了某些技术和科学术语。除非在本文中另有明 确定义，本文使用的所有其它技术和科学术语都具有本公开所属领域的一般技术人员通常理解的含义。
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“抗衡离子”是指能够启动来自如蛋白质，核酸，脂质或低聚糖等大分子的微颗粒形成的带电荷或电荷极性化的分子。带电分子是否为抗衡离子可基于以下参数经验性地确定：包括但不限于蛋白质种类、pH、离子强度、使用的有机溶剂的种类，和盐和如活性试剂的其它成分的存在。如这里提供和描述的，抗衡离子可为阴离子的或具有净负电荷或电荷极性化的基团，阳离子的或具有净正电荷或电荷极性化的基团，或两性离子的和具有负和正电荷或电荷极性化的基团。
术语“微颗粒”与“微球”可互换并指包括大分子和传送感兴趣的试剂，如药物或营养添加物到受治疗者的颗粒，其尺寸范围(平均长度，宽度或直径)为约或正好0.001微米到约或正好500微米。所述试剂可为大分子，如蛋白质、核酸、脂质或多糖，或形成微颗粒的大分子可为活性试剂如药物或营养添加物的载体。所述微颗粒也可包括合成大分子，所述大分子包括聚合物，如聚乙二醇(PEG)、聚乳酸(PLA)、聚乳酸乙醇酸共聚物(PLGA)和天然聚合物，如白蛋白、明胶、壳聚糖和葡聚糖。这里描述的“微颗粒”可包括和可制备自一种特定的天然或合成大分子，或来自不止一种类型的同样的天然或合成大分子(如不止一种类型的蛋白质)，或来自不止一种不同类型的天然或合成大分子的结合(如蛋白质和核酸或蛋白质和合成聚合物)。
这里使用的术语“微颗粒”也通常指一种并不是其生产自的全部溶液的固体形态的颗粒，尽管这里也考虑包含大分子的溶液的冷冻和/或干燥的颗粒。而是，这里使用的微颗粒通常是溶液组分的一部分的集合，其包括盐、抗衡离子、溶剂和其它成分，这些由包括但不限于沉淀、沉降、相分离和胶体形成的方法形成。
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“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”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、6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 1.31：94-106，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A1-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G Sepharose 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。
“药物组合物”表示含有一种或多种本文所述抗体药物偶联物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，所述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保持抗体活性成分的稳定性，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。
本公开中，“药物组合物”和“制剂”并不互相排斥。
本公开中所述药物组合物的溶液形式，若无特殊说明，其中的溶剂均为水。
“冻干制剂”表示液体或溶液形式的药物组合物或液体或溶液制剂经真空冷冻干燥步骤之后获得的制剂或药物组合物。
本文所用术语“约”、“大约”是指数值在由本领域一般技术人员所测定的具体值的可接受误差范围内，所述数值部分取决于怎样测量或测定(即测量体系的限度)。例如，在本领域每一次实行中“约”可意味着在1内或超过1的标准差。或者，“约”或“基本上包含”可意味着至多20％的范围。此外，特别对于生物学系统或过程而言，该术语可意味着至多一个数 量级或数值的至多5倍。除非另外说明，否则当具体值在本申请和权利要求中出现时，“约”或“基本上包含”的含义应该假定为在该具体值的可接受误差范围内。
本公开中数值为仪器测量值或仪器测量后计算值，存在一定程度的误差，一般而言，正负10％均属于合理误差范围内。当然需要考虑该数值所用之处的上下文，例如，总杂质的含量，该数值为测量后误差变化不超过正负10％，可以为正负9％、正负8％、正负7％、正负6％、正负5％、正负4％、正负3％、正负2％或正负1％，优选正负5％。
“任选”或“任选地”意味着随后所描述地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的场合。例如，“任选包含1-3个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。
“取代的”指基团中的一个或多个氢原子，优选为最多5个，更优选为1～3个氢原子彼此独立地被相应数目的取代基取代。不言而喻，取代基仅处在它们的可能的化学位置，本领域技术人员能够在不付出过多努力的情况下确定(通过实验或理论)可能或不可能的取代。例如，具有游离氢的氨基或羟基与具有不饱和(如烯属)键的碳原子结合时可能是不稳定的。
常规的药物组合物的制备见中国药典。
“给予”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“给予”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予患者内用或外用治疗剂，例如包含本公开的任一种结合化合物的组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，以诱导这类症状退化或抑制这类症状发展到任何临床可测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本公开的实施方案(例如治疗方法或制品)在缓解每个目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的患者中应当减轻目标疾病症状。
“有效量”包含足以改善或预防医学疾病的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定患者或兽医学受试者的有效量可依据以下因素而变化：例如，待 治疗的病症、患者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
“置换”是指溶解抗体蛋白或抗体药物偶联物的溶剂体系的置换，例如，使用稳定制剂的缓冲体系经物理操作方式将含抗体蛋白或抗体药物偶联物的高盐或高渗溶剂体系置换，从而使抗体蛋白或抗体药物偶联物存在于稳定制剂中。所称物理操作方式包括但不限于超滤、透析或离心后复溶。
具体实施方式
以下结合实施例进一步描述本公开，但这些实施例并非是对本公开范围的限制。本公开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，如参照冷泉港实验室出版的《抗体技术实验手册》，《分子克隆手册》；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BrukerAVANCE-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 6110A MSD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1。
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ABCR GmbH&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。
1.0M 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，25G7-Fab及ADC的制备方法已在申请号为PCT/CN2023/073057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)68.76-8.69(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)68.61-8.56(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
SEQ ID NO：13
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
BufferA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：11所示，轻链序列如SEQ ID NO：10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1.0M 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：9所示，轻链序列如SEQ ID NO：10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-BlueTM IL-4/IL-13报告基因细胞株进行评估。该细胞购自Invivogen(Cat#hkb-i1413)，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-BlueTM IL-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100μL0.3％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-4Rα hu/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-wayANOVA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-4Rα hu/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。
表8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。
实施例7：吸入干粉a-d的制备
表9处方a-d组成
制备过程：
1)按表9中的处方组成加水配制辅料溶液，加入处方量的ADC-2缓冲液(实施例6制备得到)，轻柔搅拌均匀，加入处方量的异丙醇，轻柔搅拌均匀(可根据需要用盐酸调节pH)；
2)冷冻干燥。
采用SYMPATEC粒径测定仪进行粒径测定，分散压力为4bar，粒径结果见表10。
表10吸入干粉a-c的几何粒径测定结果
将制备得到的含药吸入干粉填充至透明HPMC胶囊(3号)中，10mg装量，将胶囊样品使用COPLEY公司的NGI撞击器按照中国药典2015版四部通则0951【吸入制剂微细粒子空气动力学特性测定法】规定进行APSD测试。将APSD各级分布结果输入CITDAS version 3.10软件(COPLEY)得出FPF(＜5μm)、MMAD、GSD等值，结果见表11。
表11吸入干粉空气动力学粒径(APSD)测试结果
将样品放置于不同稳定性测试条件下，通过方法SEC(大小排阻色谱)进行药物稳定性分析，结果见表12。结果显示，处方c和d在稳定性过程中单体下降幅度最小，处方中加入柠檬酸钠使大分子性质更稳定。
表12活性成分纯度测定(SEC)结果(％)
实施例8：吸入干粉e、f、g的制备
采用与实施例7处方b相同的处方组成和制备方法，区别在于对有机溶剂的用量进行调整，如表13所示。
表13制备吸入干粉e、f、g时水与异丙醇的用量
采用SYMPATEC粒径测定仪进行粒径测定，分散压力为4bar，粒径结果见表14。
表14吸入干粉e、f、g的几何粒径测定结果
实施例9：吸入干粉h-k的制备
采用与实施例7相同的配制及冻干工艺，按照表15中的处方组成制备吸入粉末。
表15处方h～k组成
采用SYMPATEC粒径测定仪进行粒径测定，分散压力为4bar，粒径结果见表16。结果显示，药物浓度由2mg/ml提高至3.5mg/ml几何粒径变化不大。
表16吸入干粉h～k的几何粒径测定结果
空气动力学粒径测定方法与实施例7中相同，APSD结果见表17。结果显示，药物浓度在2～3.5mg/ml范围内变化对FPF、MMAD等空气动力学粒径结果影响不大。
表17吸入干粉空气动力学粒径(APSD)测试结果
实施例10：吸入干粉形态观察
采用扫描电子显微镜(SEM)对吸入干粉c和f的形态进行观察，结果见图7和8。可以看出吸入干粉c和f均是由几个纳米级别的光滑球体聚集而成。
实施例11：不同规格吸入干粉的制备
吸入干粉m(0.4mg规格)的制备：称取表18中处方量的组氨酸、柠檬酸钠和0.06mg 的海藻糖，加入水配制成辅料溶液，加入处方量的ADC-2缓冲液(实施例6制备得到)，轻柔搅拌均匀，加入处方量的异丙醇，轻柔搅拌均匀。采用与实施例7相同的冻干工艺制备冻干粉，再添加剩余处方量的海藻糖作为填充剂，填充至羟丙甲纤维素空心胶囊。
吸入干粉n(2mg规格)的制备：采用与实施例7相同的配制及冻干工艺，按照表18中的处方组成制备吸入粉末m，直接填充至羟丙甲纤维素空心胶囊。
表18处方m和n组成
空气动力学粒径测定方法与实施例7中相同，APSD结果见表19。
表19吸入干粉空气动力学粒径(APSD)测试结果

Claims (21)

  1. 一种药物组合物，包含抗体药物偶联物和抗衡离子，其中所述抗体药物偶联物具有如式I所示的结构：
    其中：
    25G7-Fab包含重链可变区和轻链可变区，所述重链可变区包含：如SEQ ID NO:1或SEQ ID NO:12所示的HCDR1，如SEQ ID NO:2所示的HCDR2，如SEQ ID NO:3所示的HCDR3，所述轻链可变区包含：如SEQ ID NO:4所示的LCDR1，如SEQ ID NO:5所示的LCDR2，如SEQ ID NO:6所示的LCDR3；
    n为1至10。
  2. 根据权利要求1所述的药物组合物，其中所述25G7-Fab具有如SEQ ID NO:8所示的重链可变区，和如SEQ ID NO:7所示的轻链可变区。
  3. 根据权利要求1或2所述的药物组合物，其中所述25G7-Fab具有如SEQ ID NO:11所示的重链，和如SEQ ID NO:10所示的轻链。
  4. 根据权利要求1-3中任一项所述的药物组合物，其中所述抗体药物偶联物的浓度以蛋白浓度计为0.1mg/mL-50mg/mL，优选0.5mg/mL-10mg/mL，更优选1mg/mL-5mg/mL。
  5. 根据权利要求1-4中任一项所述的药物组合物，其中所述抗衡离子选自硫酸盐、磷酸盐、苹果酸盐、马来酸盐、草酸盐、柠檬酸盐、琥珀酸盐、富马酸盐、衣康酸盐、氯化物和溴化物中的一种或多种，优选硫酸盐、柠檬酸盐和氯化物中的一种或多种，更优选柠檬酸钠、硫酸钠、硫酸锌、硫酸镁、硫酸钾、硫酸钙、氯化镁、氯化钙和氯化锌中的一种或多种，最优选硫酸钠和/或柠檬酸钠。
  6. 根据权利要求1-5中任一项所述的药物组合物，其中所述抗衡离子的浓度为0.01mg/mL-10mg/mL，优选0.05mg/mL-5mg/mL，更优选0.1mg/mL-2mg/mL。
  7. 根据权利要求1-6中任一项所述的药物组合物，其中所述药物组合物还包含保护剂。
  8. 根据权利要求7所述的药物组合物，其中所述保护剂选自氨基酸和/或糖。
  9. 根据权利要求8所述的药物组合物，其中所述氨基酸选自甘氨酸、组氨酸、精氨酸、赖氨酸、谷氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、色氨酸、苯丙氨酸、甲硫氨酸、天门冬氨酸中的一种或多种，更优选甘氨酸或组氨酸。
  10. 根据权利要求8或9所述的药物组合物，其中所述氨基酸的浓度为0.01mg/mL-10mg/mL，优选0.05mg/mL-5mg/mL，更优选0.1mg/mL-2mg/mL。
  11. 根据权利要求8所述的药物组合物，其中所述糖选自山梨醇、甘露醇、木糖醇、海藻糖、乳糖、果糖、麦芽糖和蔗糖中的一种或多种，优选海藻糖。
  12. 根据权利要求11所述的药物组合物，其中所述糖的浓度为0.01mg/mL-10mg/mL，优选0.05mg/mL-5mg/mL，更优选0.1mg/mL-2mg/mL。
  13. 根据权利要求1-12中任一项所述的药物组合物，其中所述药物组合物还包含有机溶剂，所述有机溶剂优选甲醇、乙醇、1-丙醇、异丙醇、叔丁醇、丁醇、丙酮、乙腈、乙酸、乙酸乙酯、甲基乙基酮、甲基叔丁基醚和二甲基亚砜中的一种或几种，更优选异丙醇。
  14. 根据权利要求13所述的药物组合物，其中所述有机溶剂的量为0.1％-50％v/v，优选0.5％-30％v/v，更优选1％-10％v/v。
  15. 根据权利要求1-14中任一项所述的药物组合物，其中所述药物组合物的pH为3.0-8.0，优选4.5-6.0。
  16. 根据权利要求1-15中任一项所述的药物组合物，包含：
    a)以蛋白浓度计，1mg/mL-5mg/mL的所述抗体药物偶联物，
    b)0.1mg/mL-2mg/mL的抗衡离子，所述抗衡离子为柠檬酸钠或硫酸钠，
    c)0.1mg/mL-2mg/mL的氨基酸，所述氨基酸为组氨酸或甘氨酸，
    d)0.1mg/mL-2mg/mL的糖，所述糖为海藻糖,
    e)1％-10％v/v的有机溶剂，所述有机溶剂为异丙醇；
    所述药物组合物的pH为4.5-6.0。
  17. 一种含抗体药物偶联物的冻干制剂，其中所述冻干制剂通过将权利要求1至16中任一项所述的药物组合物冷冻干燥获得。
  18. 一种含抗体药物偶联物的复溶溶液，其中所述复溶溶液是通过将权利要求17所述的冻干制剂复溶制备获得。
  19. 一种药物组合物，其包含如权利要求17所述的冻干制剂，以及填充剂，所述填充剂选自海藻糖、乳糖、甘露醇、葡萄糖、山梨醇、右旋糖酐中的一种或多种，优选海藻糖。
  20. 一种制品，其包括容器，该容器中装有如权利要求1至16中任一项所述的药物组合物、权利要求17所述的冻干制剂、权利要求18所述的复溶溶液或权利要求19所述的干粉制剂。
  21. 根据权利要求1至16中任一项所述的药物组合物、权利要求17所述的冻干制剂、权利要求18所述的复溶溶液、权利要求19所述的干粉制剂或权利要求20所述的制品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。
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