Classifications

• • 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
  • A61K47/6835—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
  • A61K47/6845—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a cytokine, e.g. growth factors, VEGF, TNF, a lymphokine or an interferon
• • 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
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
• • 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
  • A61K47/6835—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
  • A61K47/6849—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
• • 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
  • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
• • 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 [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/22—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2866—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/55—Fab or Fab'
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/77—Internalization into the cell
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

• the disclosure belongs to the field of medicine, in particular to a drug conjugate of glucocorticoid.
• Interleukin-4 consists of 153 amino acids with a molecular weight of about 17 kDa. Initially, IL-4 was discovered because of its ability to stimulate B cell proliferation and was named B cell stimulating factor-1 (BSF-1). IL-4, like IL-13, belongs to the type I cytokine family and has a quaternary structure composed of a hydrophobic bundle core of 4 ⁇ helices. IL-4 is secreted by TH2 cells, participates in TH2-mediated immune response, and has a wide range of biological activities, including stimulating the proliferation of T cells, mast cells, granulocytes, megakaryocytes, and erythrocytes.
• BSF-1 B cell stimulating factor-1
• IL-4 can also stimulate B cells to express major histocompatibility complex class 2 molecules.
• IL-13 and IL-4 have approximately 30% amino acid sequence homology and many similar functions. Both IL-4 and IL-13 can promote B cell proliferation and co-stimulate CD40/CD40L to induce IgM type conversion to IgE.
• IL-4 promotes the aggregation of mast cells, up-regulates the expression of high-affinity IgE receptors on mast cells and IgE low-affinity receptor CD23 (Fc ⁇ RII) on B cells, up-regulates the expression of vascular endothelial cell adhesion molecule (VCAM-1), and promotes the expression of eosinophils. , T lymphocytes, monocytes and basophils transfer. Unlike IL-13, IL-4 can promote the differentiation of naive T cells into TH2.
• IL-4 needs to bind to membrane receptors to exert biological functions.
• the human interleukin receptor (IL-4R) is a heterodimer formed by two polypeptide chains, one of which has a high affinity for IL-4, because the IL-4R ⁇ chain has a strong affinity for IL-4 in the IL-4R complex.
• the combination of -4 plays a leading role, so IL-4R ⁇ is often used instead of IL-4R in many scientific studies and reports.
• IL-4R is expressed on various cells such as human B cells, mast cells, eosinophils, basophils, macrophages/monocytes, DC cells, fibroblasts, airway epithelium and smooth muscle.
• IL-4R ⁇ can form two types of receptor complexes with other subunits, and the type I receptor composed of IL-4R ⁇ and ⁇ c is mainly expressed in hematopoietic stem cells.
• IL-4 mainly acts through the type II receptor composed of IL-4R ⁇ and IL-13R ⁇ 1.
• Type II receptors are co-receptors for IL-4 and IL-13, and IL-13 functions in combination with IL-13R ⁇ 1. Both type I receptors and type II receptors transduce signals through the Jak/STAT pathway.
• IL-4R ⁇ , ⁇ c and IL-13R ⁇ 1 bind to Jak1, Jak3 and Tyk2 respectively to activate downstream pathways.
• IL-4 and IL-13 can also pass through The insulin receptor substrate family (IRS) transduces signals and finally activates PI3-K and NF- ⁇ B in the nucleus.
• IFS insulin receptor substrate family
• Blocking IL-4R can inhibit both IL-4 and IL-13 biological functions.
• Atopic dermatitis also known as atopic dermatitis or atopic dermatitis
• AD Atopic dermatitis
• IL-4, IL-5, IL-10 and IL-13 are a common disease in dermatology. It is more common in children and adolescents.
• TH2 factors are related to the disease process of AD, and the overexpression of IL-4, IL- Mice with 13 and other TH2 factors exhibit skin protection defects and AD-like symptoms [14][15] .
• IL-13 and IL-4 also play an important role in asthma.
• Asthma is a common lung inflammatory disease characterized by airway hyperresponsiveness (AHR), mucus hypersecretion, fibrosis, and elevated IgE levels.
• Nonspecific stimuli such as cold air often Leading to increased airway hyperresponsiveness, AHR and mucus hypersecretion lead to airway obstruction, which is the main cause of death in asthma.
• TH2 factor plays an important role in the process of asthma, and IL-4 and IL-13 are overexpressed in bronchial and alveolar lavage fluid of asthmatic patients.
• IL-13 and IL-4 share some functional similarities, some studies have shown that IL-13 plays a more important role than other Th2 cytokines in the progression of asthma.
• IL-13 can promote the differentiation and fibrosis of goblet cells. Injection of recombinant IL-13 into the airways of allergen-na ⁇ ve mice resulted in airway inflammation, mucus hypersecretion, and airway hyperresponsiveness, and injection of soluble IL13R ⁇ 2 prevented AHR, mucus hypersecretion, and lung inflammation in mice happened. Injection of IL-4R ⁇ antibody in an asthma model can reduce eosinophils in AHR and alveolar lavage fluid. Research suggests that blocking IL-4R ⁇ may be effective in treating asthma.
• Glucocorticoids are also more effective drugs for the treatment of allergic diseases and inflammation.
• glucocorticoids produced in vivo such as cortisol and corticosterone
• synthetic glucocorticoids such as dexamethasone, prednisone, prednisolone, and budesonide are known. Since these glucocorticoids have a steroid structure, they are collectively referred to as steroids and are used in the treatment of various diseases. However, these steroids may cause side effects such as steroid peptic ulcer, steroid purpura, steroid pancreatitis, steroid diabetes, steroid cataract, and steroid glaucoma due to their use.
• ADC Antibody drug conjugate
• ADC refers to a monoclonal antibody or antibody fragment linked to a biologically active drug through a stable chemical linker compound.
• Most ADCs in preclinical and clinical development are used in oncology indications, where the cytotoxic payload targets antigen-expressing cancer cells.
• modulation of pathogenic cellular activity via ADC-mediated delivery of bioactive small molecules is also attractive for non-oncology indications, leading to widespread adoption of this technology.
• glucocorticoids Some drug conjugates of glucocorticoids have been disclosed in the prior art, such as WO2017210471, WO2019106609, WO2019136487, etc.
• ADC antibody-drug conjugate
• Ab is an anti-IL-4R antibody or an antigen-binding fragment thereof
• L is a linker covalently linking Ab to D
• k is 1 to 20 (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20 or any value between any two values)
• D is a glucocorticoid or a residue thereof, and the glucocorticoid is selected from dexamethasone, prednisone, prednisolone, budesonide, mometasone, beclomethasone dipropionate, fluticasone, triamcinolone acetonide and cyclic
• a sononide for example may be budesonide or ciclesonide.
• D is represented by the following formula:
• anti-IL-4R antibodies or antigen-binding fragments thereof may be known, such as anti-IL described in, for example, WO2010053751 , WO2001092340 , WO2008054606 , WO2014031610 , WO2020038454 (each of which is incorporated herein by reference) -4R antibody or antigen-binding fragment thereof.
• anti-IL-4R antibodies or antigen-binding fragments thereof include, but are not limited to, Dupixent, PRS-060, AK-120, 63 IgG1, CBP201, AMG-317, or antigen-binding fragments thereof.
• the anti-IL-4R antibody or antigen-binding fragment thereof is an anti-human IL-4R antibody or antigen-binding fragment thereof.
• the anti-IL-4R antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises:
• And/or antibody light chain variable regions comprising:
• Table 1 shows the CDR sequences of anti-IL-4R antibodies or antigen-binding fragments thereof.
• the anti-IL-4R antibody or antigen-binding fragment thereof comprises any one of the following (I) to (IV):
• a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively;
• a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively;
• Light chain variable region which comprises respectively shown in SEQ ID NO: 38, SEQ ID NO: 7 and SEQ ID NO: 40 LCDR1, LCDR2 and LCDR3;
• a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 42, SEQ ID NO: 39 and SEQ ID NO: 8, respectively.
• the anti-IL-4R antibody or antigen-binding fragment is a murine antibody, a chimeric antibody, a fully human antibody, a humanized antibody or a fragment thereof. In some specific embodiments, the anti-IL-4R antibody or antigen-binding fragment is humanized.
• the anti-IL-4R antibody or antigen-binding fragment thereof comprises a FR sequence derived from human germline light chain IGKV3-11*01 (SEQ ID NO: 22, used for antibody 25G7) or A sequence with a back mutation that is at least 95% identical to it.
• the back mutation is selected from one or more of L46P, L47W, and F71Y.
• the anti-IL-4R antibody or antigen-binding fragment thereof comprises a FR region sequence derived from human germline heavy chain IGHV3-48*01 (SEQ ID NO: 21, used for antibody 25G7) Or a sequence with back mutations at least 95% identical thereto.
• the back mutation is selected from one or more of S94A, F67S, and A93T.
• the anti-IL-4R antibody or antigen-binding fragment thereof comprises a FR region sequence derived from human germline light chain IGKV2D-29*01 (SEQ ID NO: 24, used for antibody 7B10) Or a backmutated sequence having at least 95% identity therewith.
• the back mutation is selected from M4L and/or V58I.
• the anti-IL-4R antibody or antigen-binding fragment thereof comprises a FR region sequence derived from human germline heavy chain IGHV1-2*02 (SEQ ID NO: 23, used for antibody 7B10) Or a backmutated sequence having at least 95% identity therewith.
• the back mutation is selected from one or more of M69L, R71I, T73K, and R94K.
• the anti-IL-4R antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:
• the heavy chain variable region contains:
• the heavy chain variable region of the anti-IL-4R antibody or antigen-binding fragment is shown in the sequence SEQ ID NO: 1, and the light chain variable region is shown in the sequence SEQ ID NO: 2; or
• variable region of the heavy chain is shown in the sequence SEQ ID NO: 9
• variable region of the light chain is shown in the sequence SEQ ID NO: 10; or
• variable region of the heavy chain is shown in the sequence of SEQ ID NO: 43, and the variable region of the light chain is shown in the sequence of SEQ ID NO: 37; or
• the heavy chain variable region is shown in the sequence SEQ ID NO: 43, and the light chain variable region is shown in the sequence SEQ ID NO: 41.
• the anti-IL-4R antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:
• the heavy chain variable region contains:
• the heavy chain variable region is as shown in one of the sequences of SEQ ID NO:25-27, and the light chain variable region is as shown in one of the sequences of SEQ ID NO:28-30;
• the heavy chain variable region is shown in one of the sequences SEQ ID NO: 31-33, and the light chain variable region is shown in one of the sequences SEQ ID NO: 34-36.
• the anti-IL-4R antibody or antigen-binding fragment thereof comprises a heavy chain constant region selected from human IgG1, IgG2, IgG3 or IgG4 or variants thereof. In some specific schemes, the heavy chain constant region of human IgG1 or its variants are included. In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof comprises the constant region of human ⁇ , ⁇ chain or variants thereof.
• the anti-IL-4R antibody or antigen-binding fragment thereof wherein the antibody is a humanized antibody
• the heavy chain sequence is as shown in SEQ ID NO: 17 or has at least 85% sequence identity therewith
• the light chain sequence is as set forth in SEQ ID NO: 18 or has at least 85% sequence identity thereto.
• the anti-IL-4R antibody or antigen-binding fragment thereof wherein the antibody is a humanized antibody
• the heavy chain sequence is as shown in SEQ ID NO: 19 or has at least 85% sequence identity therewith
• the light chain sequence is as set forth in SEQ ID NO: 20 or has at least 85% sequence identity thereto.
• the anti-IL-4R antibody or antigen-binding fragment thereof wherein the antibody is a humanized antibody
• the heavy chain sequence is as shown in SEQ ID NO: 44 or has at least 85% sequence identity therewith
• the light chain sequence is as set forth in SEQ ID NO: 45 or has at least 85% sequence identity thereto.
• the anti-IL-4R antibody or antigen-binding fragment thereof wherein the antibody is a humanized antibody
• the heavy chain sequence is as shown in SEQ ID NO: 44 or has at least 85% sequence identity therewith
• the light chain sequence is as set forth in SEQ ID NO: 46 or has at least 85% sequence identity thereto.
• an isolated anti-IL-4R antibody or antigen-binding fragment thereof characterized in that it competes with any anti-IL-4R antibody or antigen-binding fragment thereof as described above for binding to human IL-4R or its epitope.
• a bispecific antibody or a multispecific antibody comprising any of the above-mentioned anti-IL-4R antibodies or antigen-binding fragments thereof light chain variable region and/or heavy chain variable region .
• a single chain antibody comprising the light chain variable region and/or the heavy chain variable region of any anti-IL-4R antibody or antigen-binding fragment thereof as described above.
• the humanized anti-IL-4R antibody or antigen-binding fragment thereof further comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof.
• a human IgG2 or IgG4 heavy chain constant region is included. Because IgG2 or IgG4 has no ADCC toxicity.
• IgG1 without ADCC antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity
• the variant comprises a heavy chain constant region mutation with reduced or absent ADCC effector function, such as, but not limited to, N297A, L234A, L235A of IgGl.
• IgG1 comprises mutations E239D and M241L.
• anti-IL-4R antibodies or antigen-binding proteins thereof of the present disclosure are coded according to Kabat.
• the anti-IL-4R antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises:
• the heavy chain sequence is set forth in SEQ ID NO: 17, and the light chain sequence is set forth in SEQ ID NO: 18; or
• the heavy chain sequence is set forth in SEQ ID NO: 19, and the light chain sequence is set forth in SEQ ID NO: 20; or
• the heavy chain sequence is set forth in SEQ ID NO: 44, and the light chain sequence is set forth in SEQ ID NO: 45; or
• the heavy chain sequence is set forth in SEQ ID NO: 44, and the light chain sequence is set forth in SEQ ID NO: 46; or
• the heavy chain sequence is shown in SEQ ID NO:47, and the light chain sequence is shown in SEQ ID NO:45.
• the anti-IL-4R antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises:
• the heavy chain sequence is set forth in SEQ ID NO: 48, and the light chain sequence is set forth in SEQ ID NO: 18; or
• the heavy chain sequence is set forth in SEQ ID NO: 49, and the light chain sequence is set forth in SEQ ID NO: 18; or
• the heavy chain sequence is set forth in SEQ ID NO: 50, and the light chain sequence is set forth in SEQ ID NO: 45; or
• the heavy chain sequence is set forth in SEQ ID NO: 50, and the light chain sequence is set forth in SEQ ID NO: 46; or
• the heavy chain sequence is set forth in SEQ ID NO: 51, and the light chain sequence is set forth in SEQ ID NO: 45; or
• the heavy chain sequence is shown in SEQ ID NO:51, and the light chain sequence is shown in SEQ ID NO:46.
• the anti-IL-4R antibody or antigen-binding fragment thereof may be an antibody variant having 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid changes, and/or 1 to 10 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid changes in the heavy chain.
• the above-mentioned variant has the same or similar biological function or effect as the parental anti-IL-4R antibody or fragment thereof.
• antigen-binding fragments include, but are not limited to, Fab, Fab', Fv, F(ab')2, linear antibody, scFv (single chain Fv antibody), tandem di-scFv, tandem tri-scFv, double chain Antibody (diabody), triabody (triabody), Tetrabody, sdAb (single domain antibody or nanobody), sdFv, peptibody, domain antibody, multispecific antibody (e.g. bispecific, trispecific or tetraspecific) , dsFv (disulfide bond stabilized Fv), ScdsFv (disulfide bond stabilized single chain Fv antibody).
• the antigen-binding fragment of the anti-IL-4R antibody in the antibody-drug conjugate of the present disclosure or a pharmaceutically acceptable salt or solvate thereof is combined with the antigen of the above-mentioned anti-IL-4R antibody
• the binding fragments bind the same IL-4R or an epitope thereof.
• a polynucleotide such as DNA or RNA, encoding an anti-IL-4R antibody or antigen-binding fragment thereof as described above is provided.
• expression vectors comprising the polynucleotides described above are provided, such as eukaryotic expression vectors, prokaryotic expression vectors, viral vectors.
• host cells transformed with the above expression vectors such as eukaryotic cells and prokaryotic cells
• the host cell is bacteria (such as Escherichia coli), yeast (such as Pichia pastoris), mammalian cells (such as Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) 293 cells).
• bacteria such as Escherichia coli
• yeast such as Pichia pastoris
• mammalian cells such as Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) 293 cells.
• a method for preparing the above-mentioned anti-IL-4R antibody or antigen-binding fragment thereof comprising the steps of: expressing the antibody or antigen-binding fragment thereof in a host cell as described above, and isolating the antibody or antigen-binding fragment thereof from the host cell The antibody or antigen-binding fragment thereof.
• k is any value between 1-10, preferably any value between 2-5. k can be an integer or a decimal.
• the linker is extracellularly stable such that the ADC remains intact when present in the extracellular environment, but is capable of cleavage when internalized in the cell.
• the ADC enters a cell expressing an antigen specific for the antibody portion of the ADC
• the glucocorticoid drug moiety is cleaved from the antibody portion, and the cleavage releases the unmodified form of the glucocorticoid.
• the cleavable moiety in the linker is a cleavable peptide moiety.
• ADCs comprising a cleavable peptide moiety exhibit lower levels of aggregation, improved antibody to drug ratio relative to ADCs comprising other cleavable moieties.
• addition of a cleavable moiety increases cytotoxicity and/or potency relative to a non-cleavable linker.
• the cleavable peptide moiety is capable of being cleaved by an enzyme, and the linker is one that is cleavable by an enzyme.
• the enzyme is a cathepsin and the linker is a linker that the cathepsin is capable of cleaving.
• an enzyme-cleavable linker eg, a cathepsin-cleavable linker
• a linker comprises a stretcher unit, a chemical moiety fragment covalently linked at one end to the antibody through a carbon atom and at the other end to an amino acid unit, a disulfide moiety, a sulfonamide moiety, or a non-peptidic chemical moiety.
• Exemplary stretching units include, but are not limited to
• the linker comprises an amino acid unit, preferably comprising 2 to 7 amino acids selected from the group consisting of phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, Aspartic acid, homolysine, n-methyl-valine, (q is an integer of 1-6) amino acid residues
• exemplary amino acid units include but not limited to valine-citrulline (Val-Cit), alanine-phenylalanine (Ala-Phe ); Phenylalanine-Lysine (Phe-Lys), Phenylalanine-Homolysine (Phe-Homolys), n-Methyl-Valine-Citrulline (Me-Val-Cit) , alanine-alanine (Ala-Ala), glycine-glutamic acid (Gly-Glu), glutamic acid-alanine-alanine (Glu-Ala-Ala) and g
• a linker can comprise at least one polyethylene glycol (PEG) moiety.
• PEG polyethylene glycol
• the PEG moiety may for example contain -(PEG) p1- , where p1 is an integer from 1 to 20, for example (PEG) 2 ; (PEG) 4 ; (PEG) 5 .
• the linker comprises a spacer unit attached to D.
• the spacer unit comprises p-aminobenzyloxycarbonyl (PAB),
• the spacer unit comprises p-aminobenzoyl
• the spacer unit comprises:
• n1 is an integer selected from 0-6;
• R 7 is selected from hydrogen, C 1-6 alkyl, -(CH 2 ) n3 -COOH, -(CH 2 ) n4 -OH, n3 is selected from integers between 1-4 , n4 is selected from an integer between 1-6;
• L b represents -CR 8 (R 9 )-, -O-, -NR 10 - or a single bond, R 8 and R 9 are independently selected from hydrogen, C 1-6 alkane Group, C 3-6 cycloalkyl, -(CH 2 ) n5 -NH 2 , -(CH 2 ) n6 -COOH, -(CH 2 ) n7 -
• R 8 and R 9 in -NH(CH 2 ) n1 -L a -L b -L c - are independently selected from hydrogen, C 1-6 alkyl, C 3-6 cycloalkyl, for example Hydrogen, methyl, ethyl or cyclopropyl.
• L a represents -O- or a single bond
• L b represents -CR 8 (R 9 )- or a single bond
• R 8 and R 9 form a C 3-6 cycloalkyl group together with the carbon atoms they are connected to.
• L a represents -O- or a single bond
• L b represents -CR 8 (R 9 )- or a single bond
• R 8 and R 9 are independently selected from hydrogen, C 1-6 alkyl, C 3-6 cycloalkyl, -(CH 2 ) n5 -NH 2 , -(CH 2 ) n6 -COOH, -(CH 2 ) n7 -OH
• R 10 is selected from hydrogen or C 1-6 alkyl
• n5 is selected from integers between 0-6, n6 is selected from integers between 1-4, n7 is selected from 1 Integer between -4, and when n5 is 0, R 8 and R 9 are different.
• L a represents -O- or a single bond
• L b represents -CR 8 (R 9 )- or a single bond
• L c represents -CH 2 -.
• L a represents -O- or a single bond
• L b represents -CR 8 (R 9 )- or a single bond
• L c represents -CH 2 -
• R 8 and R 9 are independently selected from hydrogen, C 1-6 alkyl, C 3-6 cycloalkyl, preferably hydrogen, methyl, ethyl or cyclopropyl.
• -NH(CH 2 ) n1 -L a -L b -L c - in the antibody-drug conjugate is: -NHCH 2 -, -NHCH 2 CH 2 -, -NHCH 2 CH 2 CH 2 -, -NHCH 2 -O-CH 2 -, -NHCH 2 CH 2 -O-CH 2 -, -NH(CH 2 ) 3 -C(O)-, -NHCH 2 -O-CH 2 -C( O)-, -NH(CH 2 ) 2 -O-CH 2 -C(O)-,
• -NH-(CH 2 ) n1 -L a -L b -L c - is -NHCH 2 - or -NHCH 2 CH 2 CH 2 -.
• the spacer unit in the linker comprises (PEG) 2 .
• ADCs comprising shorter spacer units (eg , (PEG) 8 ) exhibit lower Aggregation levels and/or higher drug loading.
• L-D in the antibody conjugate (ADC) of the present disclosure is a chemical moiety represented by the formula:
• Str is the stretch base unit covalently linked to Ab
• Pep is selected from amino acid units, disulfide moieties, sulfonamide moieties, or the following non-peptide chemical moieties:
• W is -NH-heterocycloalkylene- or heterocycloalkyl
• Y is heteroarylene, arylene, -C(O)C 1-6 alkylene, C 2-6 alkenylene , C 1-6 alkylene or -C 1-6 alkylene-NH-
• each R 16 is independently selected from C 1-6 alkyl, C 2-6 alkenyl, -(C 1-6 alkylene Base) NHC (NH) NH 2 or - (C 1-6 alkylene) NHC (O) NH 2
• R 17 and R 18 are each independently selected from hydrogen, C 1-6 alkyl, C 2-6 alkenyl , aryl, heteroaryl, or R 17 and R 18 together can form C 3-6 cycloalkyl
• R 19 and R 20 are each independently selected from C 1-6 alkyl, C 2-6 alkenyl, aryl , heteroaryl, (C 1-6 alkyl)OCH 2 -, or R 19 and R 20 together can form a
• Str in the antibody-drug conjugate is selected from chemical moieties represented by the following formula:
• R 21 is selected from -W 1 -C(O)-, -C(O)-W 1 -C(O)-, -(CH 2 CH 2 O) p1 C(O)-, -(CH 2 CH 2 O) p1 CH 2 C(O)-, -(CH 2 CH 2 O) p1 CH 2 CH 2 C(O)-, wherein W1 is selected from C 1-6 alkylene, C 1-6 alkylene - cycloalkyl or straight chain heteroalkyl of 1 to 8 atoms, said heteroalkyl contains 1 to 3 heteroatoms selected from N, O or S, wherein said alkyl, cycloalkyl and straight chain
• the heteroalkyl groups are each independently optionally replaced by one or more selected from the group consisting of halogen, deuterium, hydroxyl, cyano, amino, C 1-6 alkyl, halogenated C 1-6 alkyl, deuterated C 1-6 alkyl , C 1-6 alkoxy and C
• L 1 is selected from -NR 22 -(CH 2 CH 2 O) p2 CH 2 CH 2 C(O)-, -NR 22 -(CH 2 CH 2 O) p2 CH 2 C(O)-, -S(CH 2 ) p2 C(O)-, -(CH 2 ) p2 C(O)- or a single bond, preferably a single bond;
• p2 is an integer from 1 to 20
• R 22 is selected from a hydrogen atom, C 1-6 alkyl, Halogenated C 1-6 alkyl or deuterated C 1-6 alkyl.
• R 21 in the antibody-drug conjugate Str is selected from C 1-6 alkylene C(O)-, -(CH 2 -CH 2 O) 2 C(O)-, - (CH 2 -CH 2 O) 2 CH 2 C(O)-, -(CH 2 -CH 2 O) 2 CH 2 CH 2 C(O)-, -(CH 2 -CH 2 O) 3 C(O )- and -( CH2 - CH2O ) 4C (O)-.
• the Pep is selected from the group consisting of valine-citrulline (Val-Cit), alanine-alanine-asparagine (Ala-Ala-Asn), glycine-glycine-lysine Acid (Gly-Gly-lys), Valine-Lysine (Val-lys), Valine-Alanine (Val-Ala), Valine-Phenylalanine (Val-Phe), or Glycine - Glycine-Phenylalanine-Glycine (Gly-Gly-Phe-Gly).
• the Sp is selected from -NHCH 2 -, -NHCH 2 CH 2 -, -NHCH 2 CH 2 CH 2 -, -NHCH 2 -O-CH 2 -, -NHCH 2 CH 2 -O -CH 2 -, -NH(CH 2 ) 3 -C(O)-, -NHCH 2 -O-CH 2 -C(O)-, -NH(CH 2 ) 2 -O-CH 2 -C(O )-, -NH(CH 2 ) 2 -O-CH 2 -C(O )-,
• the linker L in the antibody-drug conjugate comprises: maleimide-(PEG) 4 -CH 2 CH 2 C(O)-Gly-Gly-Phe-Gly, Maleimide-(PEG) 2 -Val-Cit, Maleimide-(PEG) 6 -Val-Cit, Maleimide-(PEG) 8 -Val-Cit , Maleimide-(PEG) 4 -CH 2 CH 2 C(O)-Val-lys, Maleimide-(CH 2 ) 5 -Val-Cit, Maleyl Imine-(CH 2 ) 5 -Val-lys, Maleimide-(CH 2 ) 5 -Gly-Gly-Phe-Gly, Maleimide-(PEG) 4 -CH 2 CH 2 C(O)-Gly-Gly-Phe-Gly, Maleimide-(PEG) 2 -Ala-Ala-Asn, Maleimide-(PEG) 6 -Ala-Ala
• the antibody-drug conjugate (ADC) of the present disclosure is represented by the following formula:
• k is selected from 1 to 10 and can be an integer or a decimal
• p1 is selected from 2, 4, 6 or 8
• p3 and p4 are each independently selected from 0, 1 or 2;
• k is selected from 1 to 10 and can be an integer or a decimal
• p1 is selected from 2, 4, 6 or 8
• p3 and p4 are each independently selected from 0, 1 or 2;
• the antibody-drug conjugate (ADC) of the present disclosure is selected from:
• k is selected from 1 to 10, and can be an integer or a decimal.
• the present disclosure also provides a compound represented by formula II-A or a pharmaceutically acceptable salt thereof,
• p1 is selected from 2, 4, 6 or 8, and p3 and p4 are each independently selected from 0, 1 or 2.
• the present disclosure also provides a compound represented by formula II-B or a pharmaceutically acceptable salt thereof,
• X is halogen
• p1 is selected from 2, 4, 6 or 8
• p3, p4 are each independently selected from 0, 1 or 2.
• the present disclosure also provides a pharmaceutical composition, comprising at least one aforementioned antibody-drug conjugate, and a pharmaceutically acceptable carrier, diluent or excipient.
• the unit dose of the pharmaceutical composition is 0.001 mg-1000 mg.
• the pharmaceutical composition contains 0.01%-99.99% of the aforementioned compound based on the total weight of the composition. In some embodiments, the pharmaceutical composition contains 0.1%-99.9% of the aforementioned compounds. In some embodiments, the pharmaceutical composition contains 0.5%-99.5% of the aforementioned compounds. In some embodiments, the pharmaceutical composition contains 1%-99% of the aforementioned compounds. In some embodiments, the pharmaceutical composition contains 2%-98% of the aforementioned compounds.
• the pharmaceutical composition contains 0.01%-99.99% of a pharmaceutically acceptable carrier, diluent or excipient based on the total weight of the composition. In some embodiments, the pharmaceutical composition contains 0.1%-99.9% of a pharmaceutically acceptable carrier, diluent or excipient. In some embodiments, the pharmaceutical composition contains 0.5%-99.5% of a pharmaceutically acceptable carrier, diluent or excipient. In some embodiments, the pharmaceutical composition contains 1%-99% of pharmaceutically acceptable carriers, diluents or excipients. In some embodiments, the pharmaceutical composition contains 2%-98% of a pharmaceutically acceptable carrier, diluent or excipient.
• the antibody-drug conjugates of the disclosure can be administered to a patient in a manner appropriate to the indication, eg, parenterally, topically, or by inhalation.
• the antagonist can be administered by, for example, intra-articular, intravenous, intramuscular, intralesional, intraperitoneal or subcutaneous routes, by bolus injection or continuous infusion. Local administration at the site of disease or injury is considered transdermal delivery and sustained release from the implant. Delivery by inhalation includes, for example, nasal or oral inhalation, use of a nebulizer, inhalation of the antagonist in aerosol form, and the like.
• Other options include eye drops; oral formulations, including pills, syrups, lozenges, or chewing gum; and topical formulations such as lotions, gels, sprays, and ointments.
• the present disclosure also provides an inhalable pharmaceutical composition, including the antibody-drug conjugate represented by formula (I) and a pharmaceutically acceptable carrier,
• Ab is an anti-IL-4R antibody or an antigen-binding fragment thereof
• L is a linker covalently linking Ab to D
• k is 1 to 20 (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20 or any value between any two values)
• D is a glucocorticoid or its residue.
• the Ab is an anti-IL-4R Fab fragment.
• the present disclosure also provides the use of the antibody-drug conjugate and/or the pharmaceutical composition comprising the antibody-drug conjugate in the preparation of a medicament for treating or preventing IL-4R-mediated diseases or disorders.
• the present disclosure also provides the use of the antibody-drug conjugate and/or the pharmaceutical composition comprising the antibody-drug conjugate in the preparation of a medicament for treating or preventing an immune disease or disorder.
• the disease or condition is selected from the group consisting of: asthma, nasal polyps, chronic sinusitis, allergic skin disease, eosinophilic esophagitis, chronic obstructive pulmonary disease, allergic rhinitis, arthritis, inflammatory disease, allergic reaction, Autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, and kidney disease. Asthma or allergic skin diseases are preferred.
• the present disclosure also provides a method of delivering a glucocorticoid to an IL-4R expressing cell, comprising the step of contacting the IL-4R expressing cell with the antibody-drug conjugate of the present disclosure.
• the present disclosure further provides a kit comprising the antibody-drug conjugate described in the present disclosure, or a pharmaceutical composition.
• linker refers to a chemical structural fragment or bond that is connected to a ligand at one end and a drug at the other end, and can also be connected after other linkers. Then connect with the drug.
• a joint may comprise one or more joint components.
• exemplary linker building blocks include 6-maleimidocaproyl (MC), maleimidopropionyl (MP), valine-citrulline (Val-Cit or vc), alanine-phenyl Alanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), and those derived from coupling with linker reagents: N-succinimidyl 4-(2-pyridylthio)pentanoate ( SPP), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate (SMCC, also referred to herein as MCC) and N-succinimidyl (4 -iodo-acetyl)aminobenzoate (SIAB).
• MC 6-maleimidocaproyl
• MP maleimidopropionyl
• Vcit valine-citrulline
• Linkers can include Stretch units, Spacer units, Amino Acid units and Stretcher units. It can be synthesized by methods known in the art, such as described in US2005-0238649A1.
• the linker can be a "cleavable linker" that facilitates release of the drug in the cell.
• acid-labile e.g., hydrazone
• protease-sensitive e.g., peptidase-sensitive
• photolabile, dimethyl, or disulfide-containing linkers can be used (Chari et al., Cancer Research 52:127-131 (1992); US Patent No. 5,208,020).
• stretch unit refers to a chemical structural fragment that is covalently linked to the antibody through a carbon atom at one end and to an amino acid unit, disulfide moiety, sulfonamide moiety, or non-peptidic chemical moiety at the other end.
• spacer unit is a bifunctional compound structural fragment that can be used to couple amino acid units and glucocorticoids to form antibody-drug conjugates. This coupling method can selectively link glucocorticoids to amino acid units superior.
• amino acid refers to an organic compound containing an amino group and a carboxyl group in the molecular structure, and both the amino group and the carboxyl group are directly connected to the -CH- structure.
• the general formula is H2NCHRCOOH, R is H, substituted or unsubstituted alkyl, etc. According to the amino linkage
• the positions of carbon atoms in carboxylic acids can be divided into ⁇ , ⁇ , ⁇ , ⁇ , ⁇ ...-amino acids.
• amino acids that make up natural proteins have their specific structural characteristics, that is, their amino groups are directly connected to the ⁇ -carbon atom, that is, ⁇ -amino acids, including glycine (Glycine), alanine (Alanine), valine (Valine), Leucine, Isoleucine, Phenylalanine, Tryptophan, Tyrosine, Aspartic acid, Histidine, Asparagine, Glutamic acid, Lysine, Glutamine, Methionine, Arginine , Serine, Threonine, Cysteine, Proline, etc. Unnatural amino acids such as citrulline.
• antibody-drug conjugate means that a ligand is linked to a biologically active drug through a stable linker unit.
• antibody drug conjugate refers to linking a monoclonal antibody or antibody fragment with a biologically active glucocorticoid through a stable linker unit. Wherein the antibody or antibody fragment can be combined with the glucocorticoid molecule comprising the linker through a specific group (such as an interchain disulfide bond).
• drug loading refers to the average amount of drug carried by each antibody-drug conjugate molecule in the antibody-drug conjugate population, and can also be expressed as the ratio of the amount of drug to the amount of antibody.
• the range of drug loading can be 1-20, preferably 1-10 glucocorticoids (D) linked to each antibody (Ab).
• the drug loading is expressed as k, which may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or the mean of any two values in between.
• the average amount of drug per ADC molecule after conjugation can be identified using routine methods such as UV/Vis spectroscopy, mass spectrometry, ELISA assay, monoclonal antibody size variant assay (CE-SDS) and HPLC characterization.
• the molecular size variant determination method (CE-SDS) of the disclosed monoclonal antibody can adopt the sodium dodecyl sulfate capillary electrophoresis (CE-SDS) ultraviolet detection method, under reducing and non-reducing conditions, according to the molecular weight, according to the capillary electrophoresis method (2015 edition of "Chinese Pharmacopoeia” 0542), quantitatively determine the purity of recombinant monoclonal antibody products.
• CE-SDS sodium dodecyl sulfate capillary electrophoresis
• the glucocorticoid is coupled to the N-terminal amino group of the ligand and/or the ⁇ -amino group of the lysine residue through a linking unit.
• the number of drug molecules will be less than the theoretical maximum.
• the loading of antibody-drug conjugates can be controlled by the following non-limiting methods, including:
• antibody refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds.
• the amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different.
• immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are ⁇ , ⁇ , and ⁇ chains, respectively. , ⁇ chain, and ⁇ chain.
• IgG can be divided into different subclasses according to the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds.
• IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
• Light chains are classified as either kappa chains or lambda chains by difference in the constant region.
• Each of the five Ig classes can have either a kappa chain or a lambda chain.
• variable region The sequence of about 110 amino acids near the N-terminal of the antibody heavy chain and light chain varies greatly, which is the variable region (Fv region); the rest of the amino acid sequence near the C-terminal is relatively stable, which is the constant region.
• the variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conserved sequences. Three hypervariable regions determine the specificity of antibodies, also known as complementarity determining regions (CDR).
• CDR complementarity determining regions
• Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of 3 CDR regions and 4 FR regions, and the sequence from the amino terminal to the carboxyl terminal is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
• the 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
• Antibodies of the present disclosure include murine antibodies, chimeric antibodies, humanized antibodies and fully human antibodies, preferably humanized antibodies and fully human antibodies.
• murine antibody in this disclosure refers to an antibody prepared using a mouse according to the knowledge and skill in the art. In preparation, test subjects are injected with the specified antigen, and hybridomas expressing antibodies having the desired sequence or functional properties are isolated.
• chimeric antibody is an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody.
• To establish a chimeric antibody it is necessary to first establish a hybridoma that secretes a mouse-derived specific monoclonal antibody, then clone the variable region gene from the mouse hybridoma cell, and then clone the constant region gene of the human antibody as required, and then clone the mouse variable region gene It is connected with the human constant region gene to form a chimeric gene and inserted into an expression vector, and finally expresses the chimeric antibody molecule in a eukaryotic system or a prokaryotic system.
• humanized antibody also known as CDR-grafted antibody (CDR-grafted antibody) refers to the antibody variable region framework grafted with mouse CDR sequences to humans, that is, different types of human germline antibodies Antibodies generated in the framework sequences. It can overcome the heterologous reaction induced by chimeric antibodies due to carrying a large amount of mouse protein components.
• framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.
• the germline DNA sequences of the human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk/vbase), as well as in Kabat, EA et al.
• the humanized antibody of the present disclosure also includes the humanized antibody after affinity maturation of CDR by phage display. Further descriptions of methods involving the use of mouse antibodies in humanization include, for example, Queen et al., Proc., Natl. 321, 522 (1986), Riechmann, et al., Nature, 332, 323-327 (1988), Verhoeyen, et al., Science, 239, 1534 (1988)].
• the development of monoclonal antibodies has gone through four stages, namely: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully human monoclonal antibodies.
• the present disclosure is a fully human monoclonal antibody.
• the relevant technologies for the preparation of fully human antibodies mainly include: human hybridoma technology, EBV transformed B lymphocyte technology, phage display technology (phage display), transgenic mouse antibody preparation technology (transgenic mouse) and single B cell antibody preparation technology, etc.
• antigen-binding fragment refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. It has been shown that fragments of full-length antibodies can be utilized to perform the antigen-binding function of the antibody.
• binding fragments included in "antigen-binding fragments" include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F(ab') 2 fragments, comprising (iii) Fd fragment consisting of VH and CH1 domains; (iv) Fv fragment consisting of VH and VL domains of a single arm of an antibody; (v ) a single domain or dAb fragment (Ward et al., (1989) Nature 341:544-546) consisting of a VH domain; and (vi) isolated complementarity determining regions (CDRs) or (vii) optionally via A combination of two or more isolated CDRs joined by a synthetic linker.
• CDRs complementarity
• the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked by a synthetic linker using recombinant methods, thus making it possible to produce a single protein in which the VL and VH regions pair to form a monovalent molecule. chain (referred to as single-chain Fv (scFv); see, eg, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:5879-5883).
• single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody.
• Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.
• Antibodies can be of different isotypes, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.
• Fab is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity among fragments obtained by treating an IgG antibody molecule with the protease papain (cleaving the amino acid residue at position 224 of the H chain), in which About half and the entire L chain is held together by disulfide bonds.
• F(ab')2 is an antibody having a molecular weight of about 100,000 and having antigen-binding activity and comprising two Fab regions connected at the hinge position obtained by digesting the lower portion of the two disulfide bonds in the IgG hinge region with the enzyme pepsin fragment.
• Fab' is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity obtained by cleaving the disulfide bond in the hinge region of the above-mentioned F(ab')2.
• the Fab' fragment can be produced by inserting DNA encoding a Fab' fragment of an antibody into a prokaryote expression vector or a eukaryote expression vector and introducing the vector into a prokaryote or eukaryote to express the Fab'.
• single-chain antibody single-chain Fv or “scFv” is meant to comprise an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker molecules.
• Such scFv molecules may have the general structure: NH2 -VL-linker-VH-COOH or NH2 -VH-linker-VL-COOH.
• Suitable prior art linkers consist of the repeated GGGGS amino acid sequence or variants thereof, for example using 1-4 repeat variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448) .
• linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur.J. Immuno 1.31:94-106, Hu et al. (1996) , Cancer Res. 56:3055-3061, Described by Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001 ), Cancer Immunol.
• CDR refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contribute to antigen binding.
• One of the most commonly used definitions of the six CDRs is provided by Kabat E.A. et al., (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242).
• the Kabat definition of CDRs applies only to CDR1, CDR2, and CDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) of the light chain variable domain, and to CDR1, CDR L2, and L3 of the heavy chain variable domain.
• CDR2 and CDR3 CDR H2, CDR H3 or H2, H3).
• CDR1, HCDR2, HCDR3 there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region.
• Amino acid sequence boundaries for CDRs can be determined using any of a variety of well-known schemes, including the "Kabat” numbering convention (see Kabat et al.
• the CDR amino acid residues in the heavy chain variable domain are numbered 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3);
• the CDR amino acid residues in the chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3).
• the CDR amino acid numbers in VH are 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1), 50- 52 (LCDR2) and 91-96 (LCDR3).
• the CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3) in human VH and amino acid residues 24- 34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3).
• the numbering of CDR amino acid residues in VH is approximately 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3)
• the numbering of CDR amino acid residues in VL is approximately 27-32 (CDR1 ), 50-52 (CDR2) and 89-97 (CDR3).
• the CDR regions of antibodies can be determined using the program IMGT/DomainGap Align.
• antibody framework refers to the portion of a variable domain VL or VH that serves as a scaffold for the antigen-binding loops (CDRs) of the variable domain. Essentially, it is a variable domain without CDRs.
• Binding to IL-4R refers to being able to interact with human IL-4R (or its epitope or fragment).
• the term "antigen-binding site” herein refers to a three-dimensional site recognized by an antibody or antigen-binding fragment herein.
• epitope refers to the site on an antigen to which an immunoglobulin or antibody specifically binds.
• An epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique spatial conformation (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Volume 66, G.E. Morris, Ed. (1996)).
• antibodies bind with an affinity (KD) of less than about 10 "7M , eg, about less than 10 "8M , 10 "9M or 10 " 10M or less.
• KD affinity
• nucleic acid molecule refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if the promoter or enhancer affects the transcription of the coding sequence.
• vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
• the carrier is a "plasmid” which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.
• the vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
• the vectors disclosed herein are capable of autonomous replication in the host cells into which they have been introduced (e.g., bacterial vectors and episomal mammalian vectors with a bacterial origin of replication) or can integrate into the genome of the host cell after introduction, thereby following The host genome is replicated together (eg, non-episomal mammalian vectors).
• Antigen-binding fragments can also be prepared by conventional methods.
• one or more human FR regions are added to the non-human CDR region by genetic engineering methods.
• the human FR germline sequence can be obtained from the website http://imgt.cines.fr of ImMunoGeneTics (IMGT) by comparing the IMGT human antibody variable region germline gene database and MOE software, or from Immunoglobulin Journal, 2001ISBN012441351 get.
• host cell refers to a cell into which an expression vector has been introduced.
• Host cells can include bacterial, microbial, plant or animal cells.
• Bacteria that are readily transformed include members of the enterobacteriaceae such as strains of Escherichia coli or Salmonella; the Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae.
• Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris.
• Suitable animal host cell lines include CHO (Chinese Hamster Ovary cell line) and NSO cells.
• Antibodies or antigen-binding fragments engineered in the present disclosure can be prepared and purified using conventional methods.
• cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors.
• the recombinant immunoglobulin expression vector can stably transfect CHO cells.
• mammalian expression systems lead to glycosylation of antibodies, especially at the highly conserved N-terminal site of the Fc region. Positive clones are expanded in serum-free medium in bioreactors for antibody production.
• the culture fluid from which the antibody has been secreted can be purified by conventional techniques. For example, use A or GSepharose FF columns with adjusted buffers for purification.
• Antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and aggregates can also be removed by conventional methods such as molecular sieves and ion exchange. The obtained product needs to be immediately frozen, such as -70°C, or freeze-dried.
• Amino acid sequence identity means that when the amino acid sequences are aligned and gaps are introduced as necessary to achieve the maximum percent sequence identity, and any conservative substitutions are not considered as part of the sequence identity, the difference between a first sequence and a second sequence is The percentage of amino acid residues that are identical to each other. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
• anti-IL-4R antibody or "antibody that binds IL-4R” refers to an antibody that is capable of binding IL-4R, eg, with sufficient affinity such that the antibody is useful as a therapeutic agent targeting IL-4R.
• the extent of binding of an anti-IL-4R antibody to an irrelevant non-IL-4R protein can be less than about 10% of the binding of the antibody to IL-4R as measured, for example, by radioimmunoassay (RIA).
• the antibody that binds IL-4R has a dissociation constant (Kd) ⁇ 1 ⁇ M, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, or ⁇ 0.1 nM.
• peptide refers to a compound fragment between amino acids and proteins, consisting of 2 or more amino acid molecules Peptide bonds are connected to each other and are structural and functional fragments of proteins, such as hormones and enzymes, which are essentially peptides.
• sucrose refers to a biomacromolecule composed of three elements, C, H, and O, and can be divided into monosaccharides, disaccharides, and polysaccharides.
• fluorescent probe refers to a characteristic fluorescence in the ultraviolet-visible-near-infrared region, and its fluorescence properties (excitation and emission wavelengths, intensity, lifetime and polarization, etc.) can vary with the properties of the environment, such as polarity, refractive index A class of fluorescent molecules that can be sensitively changed due to changes in viscosity, viscosity, etc., which can change one or several fluorescent properties by non-covalent interaction with nucleic acid (DNA or RNA), protein or other macromolecular structures, which can be used for research Properties and behavior of macromolecular substances.
• DNA or RNA nucleic acid
• protein or other macromolecular structures which can be used for research Properties and behavior of macromolecular substances.
• alkyl refers to a saturated aliphatic hydrocarbon group, which is a linear or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms.
• Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1 ,2-Dimethylpropyl, 2,2-Dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2- Methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3 -Dimethylbutyl, 2-ethylbutyl, 2-methylp
• alkyl groups containing 1 to 6 carbon atoms include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl , n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3 -Methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethyl Butyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl , 2,3-dimethylbutyl, etc.
• Alkyl groups may be substituted or unsubstituted, and when substituted, substituents may be substituted at any available point of attachment, said substituents being preferably one or more of the following groups independently selected from alkyl radical, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkane Oxy group, heterocycloalkoxy group, cycloalkylthio group, heterocycloalkylthio group, oxo group, carboxyl group or carboxylate group.
• heteroalkyl refers to an alkyl group containing one or more heteroatoms selected from N, O or S, wherein alkyl is as defined above.
• cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring containing 3 to 20 carbon atoms, preferably containing 3 to 12 carbon atoms, more preferably containing 3 to 6 carbon atoms.
• Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatriene Base, cyclooctyl, etc.; polycyclic cycloalkyl includes spiro ring, fused ring and bridged ring cycloalkyl. "Carbocycle” refers to the ring system in a cycloalkyl group.
• spirocycloalkyl refers to a polycyclic group of 5 to 20 membered monocyclic rings sharing one carbon atom (called a spiro atom), which may contain one or more double bonds, but none of the rings has complete conjugation The ⁇ -electron system. Preferably it is 6 to 14 yuan, more preferably 7 to 10 yuan. According to the number of spiro atoms shared between the rings, the spirocycloalkyl group can be divided into single spirocycloalkyl, double spirocycloalkyl or polyspirocycloalkyl, preferably single spirocycloalkyl and double spirocycloalkyl.
• spirocarbocycle refers to the ring system in a spirocycloalkyl.
• Non-limiting examples of spirocycloalkyl Examples include:
• fused cycloalkyl refers to a 5 to 20 membered all-carbon polycyclic group in which each ring of the system shares an adjacent pair of carbon atoms with other rings in the system, wherein one or more rings may contain one or Multiple double bonds, but none of the rings have a fully conjugated ⁇ -electron system. Preferably it is 6 to 14 yuan, more preferably 7 to 10 yuan. According to the number of rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic condensed cycloalkyl groups, preferably bicyclic or tricyclic, more preferably 5-membered/5-membered or 5-membered/6-membered bicycloalkyl groups.
• fused carbocycle refers to the ring system in a fused cycloalkyl group. Non-limiting examples of fused cycloalkyl groups include:
• bridged cycloalkyl refers to a 5 to 20 membered, all-carbon polycyclic group having any two rings sharing two carbon atoms not directly attached, which may contain one or more double bonds, but none of the rings has a complete Conjugated ⁇ -electron systems. Preferably it is 6 to 14 yuan, more preferably 7 to 10 yuan. According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic bridged cycloalkyl groups, preferably bicyclic, tricyclic or tetracyclic, more preferably bicyclic or tricyclic.
• bridged cycloalkyl groups include:
• the cycloalkyl ring may be fused to an aryl, heteroaryl or heterocycloalkyl ring where the ring bonded to the parent structure is a cycloalkyl, non-limiting examples include indanyl, tetrahydronaphthalene base, benzocycloheptyl, etc.
• Cycloalkyl groups may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , heterocycloalkylthio, oxo, carboxyl or carboxylate.
• heterocyclyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent comprising 3 to 20 ring atoms, one or more of which is selected from nitrogen, oxygen or S(O) m (where m is an integer from 0 to 2), but excluding ring portions of -OO-, -OS- or -SS-, the remaining ring atoms being carbon.
• ring atoms Preferably it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably it contains 3 to 6 ring atoms.
• Non-limiting examples of monocyclic heterocyclyl groups include pyrrolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, dihydroimidazolyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolyl, piperidine group, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc., preferably piperidinyl and pyrrolidinyl.
• Polycyclic heterocyclyls include spiro, fused and bridged heterocyclyls. "Heterocycle" refers to a ring system in a heterocyclyl group.
• spiroheterocyclyl refers to a polycyclic heterocyclic group that shares one atom (called a spiro atom) between 5 to 20-membered monocyclic rings, wherein one or more ring atoms are selected from nitrogen, oxygen or S(O ) m (wherein m is an integer from 0 to 2), the remaining ring atoms are carbon. It may contain one or more double bonds, but none of the rings has a fully conjugated pi-electron system. Preferably it is 6 to 14 yuan, more preferably 7 to 10 yuan.
• the spiroheterocyclyl can be divided into single spiroheterocyclyl, double spiroheterocyclyl or polyspiroheterocyclyl, preferably single spiroheterocyclyl and double spiroheterocyclyl. More preferably, it is a 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered or 5-membered/6-membered monospiro heterocyclic group.
• Spiroheterocycle refers to the ring system in a spiroheterocyclyl group.
• Non-limiting examples of spiroheterocyclyls include:
• fused heterocyclyl refers to a 5 to 20 membered polycyclic heterocyclic group in which each ring in the system shares an adjacent pair of atoms with other rings in the system, and one or more rings may contain one or more double bond, but none of the rings has a fully conjugated ⁇ -electron system, where one or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O) m (where m is an integer from 0 to 2), and the remaining ring
• the atom is carbon.
• it is 6 to 14 yuan, more preferably 7 to 10 yuan.
• fused heterocyclic groups preferably bicyclic or tricyclic, more preferably 5-membered/5-membered or 5-membered/6-membered bicyclic fused heterocyclic groups.
• fused heterocycle refers to a ring system within a fused heterocyclyl group.
• fused heterocyclic groups include:
• bridged heterocyclyl refers to a 5 to 14 membered polycyclic heterocyclic group in which any two rings share two atoms not directly attached, which may contain one or more double bonds, but none of the rings has a complete shared bond.
• it is 6 to 14 yuan, more preferably 7 to 10 yuan.
• bridged heterocyclyl groups include:
• the heterocyclyl ring may be fused to an aryl, heteroaryl, or cycloalkyl ring, wherein the ring attached to the parent structure is heterocyclyl, non-limiting examples of which include: wait.
• Heterocyclic groups may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alk Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , heterocycloalkylthio, oxo, carboxyl or carboxylate.
• aryl refers to a 6 to 14 membered all-carbon monocyclic or fused polycyclic (that is, rings sharing adjacent pairs of carbon atoms) group, preferably 6 to 10 membered, having a conjugated pi-electron system, such as benzene base and naphthyl.
• the aryl ring may be fused to a heteroaryl, heterocyclyl or cycloalkyl ring, where the ring bonded to the parent structure is the aryl ring.
• “Aromatic ring” refers to a ring system in an aryl group. Non-limiting examples of aryl groups include:
• Aryl groups may be substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, Alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycle Alkylthio, carboxyl or carboxylate, preferably phenyl.
• fused ring aryl can be an unsaturated aromatic condensed ring structure containing 8-14 ring atoms connected by two or more ring structures sharing two adjacent atoms.
• the number of atoms is preferably 8-12.
• it includes all unsaturated condensed ring aryl groups, such as naphthalene, phenanthrene, etc., and also includes partially saturated condensed ring aryl groups, such as benzo 3-8 membered saturated monocyclic cycloalkyl, benzo 3-8 membered partially saturated monocyclic ring alkyl.
• fused aromatic ring refers to a ring system in a fused aromatic group.
• condensed ring aryl group examples include 2,3-dihydro-1H-indenyl, 1H-indenyl, 1,2,3,4-tetrahydronaphthyl, 1,4-dihydronaphthyl and the like.
• heteroaryl refers to a heteroaromatic system comprising 1 to 4 heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen.
• Heteroaryl is preferably 5 to 12 membered, such as imidazolyl, furyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, pyrrolyl, tetrazolyl, pyridyl, pyrimidinyl, thiadiazole, pyrazine group, etc., preferably imidazolyl, pyrazolyl, pyrimidinyl or thiazolyl; more preferably pyrazolyl or thiazolyl.
• heteroaryl may be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring bonded to the parent structure is a heteroaryl ring.
• Heteroaryl refers to a ring system in a heteroaryl group.
• Non-limiting examples of heteroaryl groups include:
• Heteroaryl groups may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , heterocycloalkylthio, carboxyl or carboxylate.
• fused heteroaryl may contain 5-14 ring atoms (including at least one heteroatom), which is formed by connecting two or more ring structures with two adjacent atoms.
• Aromatic condensed ring structure, including carbon atoms, nitrogen atoms and sulfur atoms can be oxo, preferably "5-12 membered fused heteroaryl", “7-12 membered fused heteroaryl”, “9-12 membered Fused heteroaryl” etc., such as benzofuryl, benzoisofuryl, benzothienyl, indolyl, isoindole, benzoxazolyl, benzimidazole, indazolyl, benzotri Azolyl, quinolinyl, 2-quinolinone, 4-quinolinone, 1-isoquinolinone, isoquinolinyl, acridinyl, phenanthridinyl, benzopyridazinyl, phthalazinyl, quinolinyl Azolinyl
• Fused heteroaryl groups may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, Alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio group, heterocycloalkylthio group, carboxyl group or carboxylate group.
• alkylene (-CH 2 -) refers to the remaining part of an alkane molecule after removing 2 hydrogen atoms, including straight-chain and branched-chain subgroups of 1 to 20 carbon atoms.
• Alkylene groups containing 1 to 6 carbon atoms non-limiting examples include methylene (-CH 2 -), ethylene (such as -CH 2 CH 2 - or -CH(CH 3 )-), ethylene Propyl (eg -CH 2 CH 2 CH 2 - or -CH(CH 2 CH 3 )-), butylene (eg -CH 2 CH 2 CH 2 CH 2 -).
• alkylene group may be substituted or unsubstituted, and when substituted, the substituent may be substituted at any available point of attachment, preferably one or more of the following groups independently selected from halogen, deuterium, hydroxyl, Nitro, cyano or amino.
• alkenylene "heteroarylene”, “arylene”, “heterocycloalkylene” and the like are as defined above.
• alkenylene refers to a group comprising linear alkenes having 2 to 8 carbon atoms, preferably 2 to 6 carbon atoms, more preferably 2 to 4 carbon atoms, and having at least one double bond in any position. groups, including, for example, vinylidene, allylene, propenylene, butenylene, prenylene, butadienylene, pentenylene, pentylene Alkenyl, hexenylene, hexadienylene, etc.
• alkynylene includes linear alkynylene groups having 2 to 8 carbon atoms, preferably 2 to 6 carbon atoms, more preferably 2 to 4 carbon atoms, and having at least one triple bond in any position. groups, including, for example, ethynylene, propynylene, butynylene, pentynylene, hexynylene, and the like.
• alkoxy refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl is as defined above.
• alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy.
• Alkoxy may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkoxy Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , heterocycle Alkylthio, carboxyl or carboxylate.
• the substituent is preferably one or more of the following groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkoxy Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycl
• alkylthio refers to -S-(alkyl) and -S-(unsubstituted cycloalkyl), wherein alkyl is as defined above.
• alkylthio include: methylthio, ethylthio, propylthio, butylthio, cyclopropylthio, cyclobutylthio, cyclopentylthio, cyclohexylthio.
• Alkylthio groups may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alk Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , One or more substituents in heterocycloalkylthio.
• hydroxyalkyl refers to an alkyl group substituted by a hydroxy group, wherein alkyl is as defined above.
• haloalkyl refers to an alkyl group substituted with a halogen, wherein alkyl is as defined above.
• deuteroalkyl refers to an alkyl group substituted with a deuterium atom, wherein alkyl is as defined above.
• hydroxyl refers to a -OH group.
• a carbon atom is linked to an oxygen atom by a double bond, where a ketone or aldehyde group is formed.
• a carbon atom is double bonded to a sulfur atom to form a thiocarbonyl group -C(S)-.
• halogen refers to fluorine, chlorine, bromine or iodine.
• amino refers to -NH2 .
• cyano refers to -CN.
• nitro refers to -NO2 .
• aldehyde refers to -CHO.
• carboxylate refers to -C(O)O(alkyl) or -C(O)O(cycloalkyl), wherein alkyl and cycloalkyl are as defined above.
• acyl halide refers to a compound containing the group -C(O)-halogen.
• sulfonyl refers to -S(O)(O)-.
• amino-protecting group is a suitable group known in the art for amino-protection, referring to the amino-protecting group in the literature ("Protective Groups in Organic Synthesis", 5 Th.Ed.TW Greene & P.GMWuts), preferably , the amino protecting group can be (C 1-10 alkyl or aryl) acyl, for example: formyl, acetyl, benzoyl, etc.; can be (C 1-6 alkyl or C 6-10 aryl base) sulfonyl; it can also be (C 1-6 alkoxy or C 6-10 aryloxy) carbonyl, for example: Boc or Cbz; it can also be substituted or unsubstituted alkyl, for example: trityl 2,4-dimethoxybenzyl (DMB), p-methoxybenzyl (PMB) or benzyl (Bn).
• DMB trityl 2,4-dimethoxybenzyl
• PMB
• Optional or “optionally” means that the subsequently described event or circumstance can but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur.
• a heterocycloalkane group optionally substituted with an alkyl group means that an alkyl group may but need not be present, and the specification includes cases where the heterocycloalkane group is substituted with an alkyl group and where the heterocycloalkane group is not substituted with an alkyl group. situation of replacement.
• pharmaceutical composition means a mixture containing one or more compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, as well as other components such as physiologically/pharmaceutically acceptable Carriers and Excipients.
• the purpose of the pharmaceutical composition is to promote the administration to the organism, facilitate the absorption of the active ingredient and thus exert biological activity.
• drug carrier used in the drug of the present disclosure refers to a system that can change the way the drug enters the human body and distributes the drug in the body, controls the release rate of the drug, and delivers the drug to the target organ.
• Drug carrier release and targeting system enables reduction of drug Degradation and loss, reduce side effects, improve bioavailability.
• polymer surfactants that can be used as carriers can self-assemble and form various forms of aggregates due to their unique amphiphilic structure.
• Preferred examples are micelles, microemulsions, gels, liquid crystals, vesicles, etc. . These aggregates have the ability to entrap drug molecules, and at the same time have good permeability to the membrane, which can be used as excellent drug carriers.
• excipient is an add-on in a pharmaceutical preparation other than the main drug, and can also be called an adjuvant.
• adjuvant such as binders, fillers, disintegrants, lubricants in tablets; matrix parts in semi-solid ointments and creams; preservatives, antioxidants, flavoring agents, fragrances, Solubilizers, emulsifiers, solubilizers, osmotic pressure regulators, colorants, etc. can all be called excipients.
• the term "diluent” is also known as filler and its main purpose is to increase the weight and volume of the tablet. The addition of the diluent not only ensures a certain volume, but also reduces the dose deviation of the main components and improves the compression molding properties of the drug. When the drug in the tablet contains oily components, an absorbent needs to be added to absorb the oily substance, so as to keep it in a "dry” state, so as to facilitate making tablets.
• Compounds in the present disclosure may contain one or more asymmetric centers and thus may give rise to enantiomers, diastereoisomers, and which may be defined as (R)- or (S)-or according to absolute stereochemistry Other stereoisomeric forms of amino acids (D)- or (L)-.
• This disclosure includes all possible isomers as well as their racemic and optically pure forms.
• Optically active (+) and (-), (R)- and (S)- or (D)- and (L)-isomers can be prepared using chiral synthons or chiral reagents, or can be prepared using conventional methods Examples include chromatography and fractional crystallization.
• the bond Indicates unassigned configuration, i.e. if chiral isomers exist in the chemical structure, the bond can be or or both and Two configurations.
• the bond If the configuration is not specified, it can be Z configuration or E configuration, or both configurations.
• Stepoisomer refers to compounds composed of the same atoms bonded by the same bonds but with different three-dimensional structures, which are not interchangeable.
• Various stereoisomers and mixtures thereof are contemplated in this disclosure and include “enantiomers,” which refer to two stereoisomers whose molecules are non-superimposable mirror images of each other.
• Tautomer refers to the transfer of a proton from one atom of a molecule to another atom of the same molecule. Tautomers of any of the described compounds are included in this disclosure.
• Atoms capable of being isotopically labeled include, but are not limited to, hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, iodine, and the like. They can be replaced by isotopes 2 H(D), 3 H, 11 C, 13 C, 14 C, 15 N, 18 F, 31 P, 32 P, 35 S, 36 Cl and 125 I, respectively.
• deuterium when a position is specifically designated as deuterium (D), the position is understood to mean deuterium having an abundance of at least 3000 times greater than the natural abundance of deuterium (which is 0.015%) (ie, at least 45 % deuterium incorporation).
• Figure 1 Dose-response curve of the whole-cell binding activity of the test protein to human IL-4R ⁇ -expressing cells
• FIG. 4 Time-concentration curves of ADC-2 and free Budesonide in various tissues after intratracheal administration
• NMR shifts ( ⁇ ) are given in units of 10 -6 (ppm).
• the determination of NMR is to use Bruker AVANCE-400 nuclear magnetic instrument, and the determination solvent is deuterated dimethyl sulfoxide (DMSO-d 6 ), deuterated chloroform (CDCl 3 ), deuterated methanol (CD 3 OD), and the internal standard is four Methylsilane (TMS).
• DMSO-d 6 dimethyl sulfoxide
• CDCl 3 deuterated chloroform
• CD 3 OD deuterated methanol
• TMS Methylsilane
• MS was determined by Shimadzu 2010 Mass Spectrometer or Agilent 6110AMSD mass spectrometer.
• HPLC uses Shimadzu LC-20A systems, Shimadzu LC-2010HT series or Agilent Agilent 1200 LC high pressure liquid chromatography (Ultimate XB-C18 3.0*150mm column or Xtimate C18 2.1*30mm column).
• Chiralpak IC-3 100 ⁇ 4.6mm I.D., 3um, Chiralpak AD-3 150 ⁇ 4.6mm I.D., 3um, Chiralpak AD-3 50 ⁇ 4.6mm I.D., 3um, Chiralpak AS-3 150 ⁇ 4.6mm were used for chiral HPLC analysis and determination I.D., 3um, Chiralpak AS-3 100 ⁇ 4.6mm I.D., 3 ⁇ m, ChiralCel OD-3 150 ⁇ 4.6mm I.D., 3um, Chiralcel OD-3 100 ⁇ 4.6mm I.D., 3 ⁇ m, ChiralCel OJ-H 150 ⁇ 4.6mm I.D., 5um, Chiralcel OJ-3 150 ⁇ 4.6mm I.D., 3um chromatographic column; use Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate for thin-layer chromatography silica gel plate, and the specification of silica gel plate used for thin-layer chromatography (TLC) is 0.15mm ⁇ 0.2mm, and the specifications
• the chiral preparative column uses DAICEL CHIRALPAK IC (250mm*30mm, 10um) or Phenomenex-Amylose-1 (250mm*30mm, 5um).
• the CombiFlash rapid preparation instrument uses Combiflash Rf150 (TELEDYNE ISCO).
• the known starting materials of the present disclosure can be adopted or synthesized according to methods known in the art, or can be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Shaoyuan Chemical Technology (Accela ChemBio Inc), Darui Chemical products and other companies.
• the reactions can all be carried out under an argon atmosphere or a nitrogen atmosphere.
• Argon atmosphere or nitrogen atmosphere means that the reaction bottle is connected to an argon or nitrogen balloon with a volume of about 1 L.
• the hydrogen atmosphere means that the reaction bottle is connected to a hydrogen balloon with a capacity of about 1L.
• the pressurized hydrogenation reaction uses Parr 3916EKX hydrogenator and Qinglan QL-500 hydrogen generator or HC2-SS hydrogen Chemometer.
• the hydrogenation reaction is usually vacuumized and filled with hydrogen, and the operation is repeated 3 times.
• the solution refers to an aqueous solution.
• reaction temperature is room temperature, which is 20°C to 30°C.
• the monitoring of the reaction process in the embodiment adopts thin-layer chromatography (TLC), the developer used for reaction, the eluent system of the column chromatography that purifies compound adopts and the developer system of thin-layer chromatography comprise: A: Dichloromethane/methanol system, B: n-hexane/ethyl acetate system, C: petroleum ether/ethyl acetate system, D: petroleum ether/ethyl acetate/methanol, the volume ratio of the solvent is determined according to the polarity of the compound Adjustment can also be adjusted by adding a small amount of alkaline or acidic reagents such as triethylamine and acetic acid.
• TLC thin-layer chromatography
• DAST diethylaminosulfur trifluoride
• THF tetrahydrofuran
• NMP N-methylpyrrolidone
• DCM dichloromethane
• m-CPBA m-chloroperoxybenzoic acid
• DIEA N,N-diisopropyl Ethylamine
• TEA triethylamine
• Boc tert-butoxycarbonyl, MeOH: methanol
• Et 2 O ether.
• Example 1 N-((10S)-10-benzyl-1-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxyl-6a,8a-dimethyl-4 -Oxo-10-propyl-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-Dodecahydro-8bH-naphtho[2',1':4, 5] Indeno[1,2-d][1,3]dioxol-8b-yl)-1,6,9,12,15-pentaoxo-3-oxo-5,8,11 , Preparation of 14-tetrapolyethylene glycol-16-yl)-1-(2-bromoacetamido)-3,6,9,12-tetraoxopentadecane-15-amide (compound 1)
• Step 5 (9H-fluoren-9-yl)methyl(2-(((2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-di Methyl-4-oxo-10-propyl-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1 ':4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethoxy)methyl)amino)-2-oxoethyl base) preparation of carbamate (compound 1f)
• Step 7) (9H-fluoren-9-yl)methyl((10S)-10-benzyl-1-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxyl-6a ,8a-Dimethyl-4-oxo-10-propyl-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[ 2',1':4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-1,6,9,12,15,18-hexaoxo Preparation of -3,21,24,27,30-pentoxo-5,8,11,14,17-pentaaminotriaconate-32-yl)carbamate (compound 1h)
• Step 9) N-((10S)-10-benzyl-1-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4- Oxo-10-propyl-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5 ]indeno[1,2-d][1,3]dioxol-8b-yl)-1,6,9,12,15-pentaoxo-3-oxo-5,8,11, Preparation of 14-tetrapolyethylene glycol-16-yl)-1-(2-bromoacetamido)-3,6,9,12-tetraoxopentadecane-15-amide (compound 1)
• Step 2) (9H-fluoren-9-yl)methyl-((10S)-10-benzyl-1-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxyl- 6a,8a-Dimethyl-4-oxo-10-propyl-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-Dodecahydro-8bH-naphtho [2',1':4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-1,6,9,12,15-pentaoxo- Preparation of 3-oxo-5,8,11,14-tetrapolyethylene glycol-16-yl)carbamate (compound 2b)
• the gene sequence of the synthetic antibody was subcloned into the pcDNA3.4 vector.
• the ligation product was transformed into Top10 competent cells, and the positive clones were picked and expanded for culture.
• the clones were inoculated in the culture medium to expand the culture, and a large number of plasmids containing the deoxyribose nucleic acid sequence of the 25G7-Fab antibody were extracted.
• liposome transfection the expression plasmid was mixed with the transfection reagent, added to Expi 293 cells, cultured in a constant temperature incubator for 5 days, and the cell culture was harvested. After the cell culture was centrifuged, the supernatant was collected, the cell debris was removed by filtration, and the clarified liquid was collected.
• AZD1402-TAG the protein sequence is from SEQ ID NO1 in patent WO2020200960A1, in order to facilitate protein Preparation and purification, the C-terminus of the sequence was fused with a His tag, and finally named AZD1402-TAG).
• the coding gene sequence of the above protein was synthesized and subcloned into pcDNA3.4. After mixing the expression plasmid and the transfection reagent, incubate at 37°C for 15 minutes, add the mixture dropwise into the HEK293 cell fluid, and culture the cell fluid on a shaker at 37°C for one week, centrifuge the cell culture and take the supernatant. Use a buffer without imidazole to equilibrate the nickel affinity chromatography column, then pass the protein sample through the nickel affinity chromatography column for loading, and again equilibrate with the buffer without imidazole, then use the buffer containing high concentration of imidazole Elute column-bound protein. The eluted protein was transferred to a dialysis bag, dialyzed in 1 ⁇ PBS, and replaced with PBS storage buffer. After detection, the target protein was obtained.
• Buffer A In a 2.0L container, add KH 2 PO 4 (8.50g), K 2 HPO 4 (8.56g), NaCl (5.86g) and EDTA (1.50g), add 1.6L water for injection, stir Half an hour, after completely dissolving, dilute to 2.0L with water for injection, and measure the pH to be 6.30 ⁇ 0.1.
• TCEP tris(2-carboxyethyl)phosphine
• TCEP tris(2-carboxyethyl)phosphine
• Test Example 1 Affinity detection between antibody drug conjugate ADC-2 and human IL-4R ⁇ protein
• the flow rate was set at 30 ⁇ L/min.
• the detection time of ADC-2, 25G7-Fab and AZD1402-TAG was 100 seconds for binding and 100 seconds for dissociation. 600 seconds.
• Biacore 8K evaluation software (Cytiva) was used for analysis to obtain the affinity data of each test protein.
• Test Example 2 Whole-cell Binding Activity of Antibody Drug Conjugate ADC-2 to Human IL-4R ⁇ Expressing Cells
• Antibody drug conjugates ADC-2, 25G7-Fab, hu25G7 (heavy chain sequence shown in SEQ ID NO: 44, light chain sequence shown in SEQ ID NO: 45), AZD1402-TAG.
• TF-1 and Karpas 299 cell lines were identified to express IL-4R ⁇ . After the TF-1 and Karpas299 cells in the logarithmic growth phase were collected, the cell density was counted and adjusted, and placed in a round-bottom 96-well plate at 1 ⁇ 10 5 cells/well. Cells were incubated with different concentrations of anti-IL-4R antibodies (25G7, naked anti-25G7-Fab), antibody-drug conjugates ADC-2 and AZD1402-TAG at 4°C for 45 minutes, and FACS Buffer (containing 2% FBS After washing away the antibody, fluorescently labeled secondary antibody was added for staining.
• anti-IL-4R antibodies 25G7, naked anti-25G7-Fab
• ADC-2 and AZD1402-TAG antibody-drug conjugates ADC-2 and AZD1402-TAG
• 25G7-Fab, hu25G7 and ADC-2 were stained with FITC-coupled anti-human IgG (Fab specific) secondary antibody (Sigma, CAT#F5512-1ML), AZD1402-TAG uses FITC-conjugated anti-His antibody (GenScript, CAT#A01620).
• the cells were incubated with the secondary antibody at 4°C for 30 minutes, then washed twice with FACS Buffer, detected by flow cytometry (BD, FACS Celesta), and the detection results were analyzed by FlowJo (FlowJo, LLC) software.
• Table 2 The EC50 value of the whole cell binding activity of the test protein to human IL-4R ⁇ expressing cells
• Test Example 3 Blocking Effect of Antibody Drug Conjugate ADC-2 on IL-4/IL-13 Signaling Pathway
• the activation of STAT6 is a key step in the activation of IL-4/IL-13 signaling pathway.
• the blocking effect of ADC-2 on IL-4/IL-13 signaling pathway was evaluated by HEK-Blue TM IL-4/IL-13 reporter gene cell line.
• the cells were purchased from Invivogen (Cat#hkb-il413), in which the human STAT6 gene and the secreted alkaline phosphatase reporter gene (Secreted alkaline phos-phatase, SEAP) induced by phosphorylated STAT6 were overexpressed.
• SEAP substrate QUANTI-Blue was used to detect the secreted SEAP content in the cell culture supernatant to assess the activation level of the IL-4/IL-13 signaling pathway.
• each of the tested proteins can block the activation of STAT6 induced by recombinant human IL-4 and IL-13, and the IC50 value of ADC-2 for blocking the IL-4 signaling pathway is 0.68nM, which is close to the IC 50 value of 0.61nM of 25G7-Fab; the IC 50 value of ADC-2 blocking IL-13 signaling pathway is 10.60nM, which is comparable to the IC 50 value of 25G7-Fab of 14.59nM.
• Test Example 4 Endocytic Activity of Antibody Drug Conjugate ADC-2 in TF-1 Cells
• Antibody drug conjugates ADC-2, 25G7-Fab, hu25G7, AZD1402-TAG.
• Collect well-growing TF-1 cells and adjust the density to 1 ⁇ 10 6 cells/mL add 100 ⁇ L/well into a 96-well cell culture plate and place at 4°C, add each test protein at a final concentration of 2 nM, and After incubating at 4°C for 1 hour, put it into the cell culture incubator at 4°C for 1 minute, and continue to cultivate under the condition of 5% CO 2 , set up a culture plate at each time point, take out the culture plate according to different incubation times, wash with FACS buffer and centrifuge , followed by incubation with fluorescently-conjugated secondary antibodies.
• 25G7-Fab, hu25G7 and ADC-2 were labeled with FITC-conjugated anti-human IgG (Fab-specific) secondary antibody, and AZD1402-TAG was labeled with FITC-conjugated anti-His antibody.
• the cells were incubated with the secondary antibody at 4°C for 30 minutes, then washed twice with FACS Buffer, detected by flow cytometry (BD, FACS Celesta), and the detection results were analyzed by FlowJo (FlowJo, LLC) software.
• the formula for calculating the endocytosis rate at a specific time point is: (gMFI Test Ab at time X -gMFI ISO Ab at time X )/(gMFI Test Ab at time zero -gMFI ISO Ab at time zero )*100%
• Test Example 5 Pharmacokinetic test of antibody drug conjugate ADC-2 in mouse model
• mice received intratracheal atomized administration (intratracheal, i.t.), and 300-400 ⁇ L of jaw blood was collected at different time points after administration, and after standing for 2 hours, centrifuged at 7500 rpm and 4°C for 10 min, serum was collected. After blood collection, the mice were anesthetized and fixed, and the neck muscles of the mice were bluntly dissected to expose the trachea. The trachea was opened laterally, and the lavage needle was slowly inserted, and the pre-pumped 4°C pre-cooled saline was slowly pushed in. Lung tissue was lavaged, and alveolar lavage fluid (BALF) was collected.
• intratracheal intratracheal, i.t.
• Blocking add 300 ⁇ L of PBS containing 3% BSA to each well, shake the plate at room temperature at 400 rpm and incubate for 2 hours.
• the detection antibody (Invitrogen, A18811) was diluted 10,000 times with Assay buffer (0.05% PBST containing 0.5% BSA), added to the ELISA plate, 100 ⁇ L per well, and incubated for 1 h at room temperature at 400 rpm.
• Plate washing add 300 ⁇ L 0.05% PBST to each well, and wash the plate 3 times.
• Termination 100 ⁇ L of substrate reaction termination solution (Solarbio, C1058) was added to each well, and the light absorption value at a wavelength of 620 nm was detected.
• the pharmacokinetic parameters of the antibody-drug conjugate ADC-2 and free Budesonide are shown in Table 6, and the drug-time curves are shown in FIG. 4 .
• Test Example 6 In vivo efficacy of ADC-2 on OVA-induced mouse asthma model
• mice were randomly divided into normal control animals and model animals. On Day 0, 7 and 14, 100 ⁇ L of PBS solution containing 200 ⁇ g OVA (Sigma, A5503) was injected intraperitoneally, and mice were sensitized with an equal volume of aluminum hydroxide as an adjuvant. On Day 21-27, the mice inhaled the nebulized solution of 3% OVA once a day for 35 minutes each time. Beginning on Day 21, half an hour before inhaling the OVA nebulized solution, the mice received intratracheal nebulized administration. The specific dosing regimen is shown in Table 7.
• the number of white blood cells and typed cells in BALF The number of white blood cells and typed cells in BALF.
• the experimental results are shown in Figure 5.
• the antibody drug conjugate ADC-2 significantly reduced the number of white blood cells, eosinophils and neutrophils in the BALF, and also had a certain effect on the number of monocytes.
• the efficacy is better than AZD1402-TAG, and significantly better than 25G7-Fab and Budesonide.
• ADC-2 shows the synergistic effect of 25G7-Fab and Budesonide in terms of drug efficacy.
• Test Example 7 Dose exploration of ADC-2 in vivo efficacy in OVA-induced mouse asthma model
• the administration and BALF sampling operations are the same as in Test Example 6.
• the specific dosage regimen is shown in Table 8.
• the number of white blood cells and typed cells in BALF The number of white blood cells and typed cells in BALF.

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