WO2012169741A2 - Anticorps humain spécifique de fcεri et composition le contenant pour traiter ou diagnostiquer des maladies allergiques - Google Patents

Anticorps humain spécifique de fcεri et composition le contenant pour traiter ou diagnostiquer des maladies allergiques Download PDF

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WO2012169741A2
WO2012169741A2 PCT/KR2012/004247 KR2012004247W WO2012169741A2 WO 2012169741 A2 WO2012169741 A2 WO 2012169741A2 KR 2012004247 W KR2012004247 W KR 2012004247W WO 2012169741 A2 WO2012169741 A2 WO 2012169741A2
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seq
chain variable
variable region
light chain
heavy chain
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WO2012169741A3 (fr
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박병덕
윤선주
배종환
박은혜
김욱동
김민희
강현정
유경숙
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(주)네오팜
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Priority claimed from KR1020120057081A external-priority patent/KR20120135868A/ko
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against Fc-receptors, e.g. CD16, CD32, CD64
    • 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
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • 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 present invention relates to a human antibody specific for Fc ⁇ RI, a receptor having high affinity for Fc of IgE, and a method for obtaining the same, wherein the Fc ⁇ RI specific antibody according to the present invention efficiently inhibits binding between IgE and Fc ⁇ RI, By preventing the signaling caused by the IgE-mediated allergic rhinitis, asthma, atopic dermatitis, urticaria and contact dermatitis, such as allergic diseases can be utilized for the treatment or diagnosis.
  • Allergic reactions that cause hypersensitivity to certain foods or drugs include allergic diseases such as allergic rhinitis, asthma, atopic skin disease, urticaria and contact dermatitis, which have a form of an immediate response in the body. Immediate response in allergic reactions is mediated by immunoglobulin E (IgE). Most allergic reactions are caused by the binding of IgE to the surface of the allergic receptor, one of the receptors for the Fc portion (Fc ⁇ ) of IgE is Fc ⁇ RI.
  • IgE immunoglobulin E
  • IgE is a major factor involved in the immediate response to allergies and is involved in the final stages of the response. IgE is mainly synthesized in B lymphocytes, and the produced IgE binds to the Fc ⁇ RI receptor with high affinity. Fc ⁇ RI is a glycoprotein molecule and plays an important role in allergic reactions. In addition, Fc ⁇ RI is expressed in mast cells, neutrophil cells, and coral cells, as well as in immune cells, dendritic cells (Allergy. 1995, 50, 193-199, Clin Exp Allergy. 1996, 26, 648- 655).
  • Human Fc ⁇ RI has three different subunits: alpha chain, beta chain (signal amplification factor) and gamma chain (signal transfer factor).
  • Tetramer Fc ⁇ RI composed of one alpha chain and one beta chain and two gamma chains, respectively, is mainly expressed on the cell membranes of tissue cells and basophils and plays an important role in type I allergic responses for cell activation. Only the alpha chain of Fc ⁇ RI binds directly to IgE and the binding site for IgE spans the extracellular region of the alpha chain (Nature. 2000, 406, 259-266).
  • Fc ⁇ RI to which IgE binds has an ⁇ -subunit (Fc ⁇ RI ⁇ ), and Fc ⁇ RI ⁇ has a size of about 60 kDa, and is composed of a hydrophobic domain existing in the cell membrane and a hydrophilic domain existing outside the cell membrane.
  • IgE bound to Fc ⁇ RI remains bound to the cell membrane for several weeks.
  • Signal transduction mechanisms are activated by the binding of allergens and the binding of Fc ⁇ RI to activate cells. This result in induces degranulation of cells.
  • Pharmacological mediators, histamine (substances that respond late to hypersensitivity reactions) and serotonin are released, causing type I allergic reactions. These substances, produced in mast cells and basophils, cause a variety of physiological allergic diseases. IgE specific for allergens continues to be expressed by B cells.
  • Fc ⁇ RI-expressing cells are known to increase in the blood of allergic patients, and the expression of Fc ⁇ RI in the blood of allergic rhinitis, atopic, asthma and atopic dermatitis patients is increased.
  • IgE is not expressed in basophils and mast cells, IgE and Fc ⁇ RI bind in a very strong and safe form. This fact indicates that IgE is always around these cells.
  • allergen avoidance therapy, antiallergic drug use, and immunotherapy for certain allergens have been used for the treatment of allergic diseases (Clin. Immunol. 1997, 100 (2), 171-176, Allergy 1998, 53, 821-). 832, Allergy Clin. Immunol. 1998, 102, 631-636, Clin.Exp. Allergy. 1999, 29 (11), 1563-1571, Allergy Clin. Immunol. 2002, 110, 154-159).
  • antihistamines and anti-inflammatory drugs of the corticosteroid system are widely used.
  • Many allergens have been developed over the past few years, but only alleviate the symptoms, and there are no treatments that can cure the root cause of allergies.
  • the efficacy of the drug is insufficient or has a number of disadvantages due to side effects. Therefore, the demand for a new concept of allergy treatment is increasing.
  • Omalizumab (Xolair) is used as a treatment for asthma patients.
  • Omalizumap binds to IgE and inhibits the release of chemicals, a major cause of asthma and allergies.
  • Omalizumab has side effects such as angioedema, anaphylactic reactions and other allergic conditions, and allergic bronchial spasms, and allergic granulomatous vasculitis and idiopathic severe thrombocytopenia have been reported through postmarketing results.
  • the present invention provides an antibody having the ability to specifically bind to Fc ⁇ RI and a method for producing such an antibody, in order to provide a therapeutic agent that can effectively and fundamentally treat allergic diseases without side effects.
  • Still another object of the present invention is to provide a pharmaceutical composition for treating allergic diseases such as allergic rhinitis, asthma, atopic skin disease, urticaria and contact dermatitis, containing the Fc ⁇ RI specific antibody.
  • Still another object of the present invention is to provide a method for suppressing allergic diseases such as allergic rhinitis, asthma, atopic skin disease, urticaria and contact dermatitis using the Fc ⁇ RI specific antibody.
  • Another object of the present invention is to provide a therapeutic agent that can be used to diagnose or treat allergic diseases, particularly allergic rhinitis, asthma, atopic skin disease, urticaria and contact dermatitis, or to treat patients with the Fc ⁇ RI specific antibody.
  • a phage display technique from a single-chain Fv (scFv) phage library to obtain a fully human antibody that binds to the Fc ⁇ RI with high affinity and specificity.
  • the preparation and phage display of the phage library can be carried out by manufacturing as known in the US Patent No. 7063943, US Patent No. 6172197 and the like.
  • the antibody according to the present invention obtained and provided by the above method comprises an Fc ⁇ RI comprising CDR1, CDR2 and CDR3 included in any one amino acid sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 and 16 CDR1, CDR2 included in any of the heavy chain variable regions of the specific antibody and in any amino acid sequence selected from SEQ ID NOs: 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40 And the light chain variable region of an Fc ⁇ RI-specific antibody comprising CDR3.
  • a light chain variable region comprising a heavy chain variable region comprising CDR1, CDR2 and CDR3 included in the heavy chain variable region of SEQ ID NO: 12 and CDR1, CDR2 and CDR3 included in the light chain variable region of SEQ ID NO: 28;
  • the fragment of the antibody according to the present invention is a single chain antibody, diabodies, triabodies, tetrabodies, Fab fragments, F (ab) having a binding function to Fc ⁇ RI ') 2 fragments, Fd, scFv, domain antibodies, bispecific antibodies, minibodies, caps, IgD antibodies, IgE antibodies, IgM antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, IgG4 antibodies, derivatives of antibody constant regions, Phosphorus receptors based on protein scaffolds, and the like, but are not limited thereto.
  • fragments of the antibody according to the present invention may exhibit the same characteristics as the antibody according to the present invention. It is obvious to those skilled in the art.
  • antibodies in which the mutation occurs in the variable region are included in the scope of the present invention.
  • One such example is conservative substitution of amino acids in the variable region.
  • Conservative substitutions mean substitutions with other amino acid residues that have properties similar to those of the original amino acid sequence. For example, lysine, arginine, and histidine have similar properties because they have a base side chain. Aspartic acid and glutamic acid It has similar characteristics in that it has a mountain side chain.
  • glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar characteristics in that they have a non-charged polar side chain, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine are nonpolar.
  • Tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they have side chains.
  • the antibody or fragment thereof according to the present invention is also available in the form of a conjugate conjugated with another substance.
  • the material that can be used by conjugation with the antibody or fragment thereof according to the present invention include, but are not limited to, an antibody for treating allergic diseases such as omalizumab, and a conventional allergic disease therapeutic agent such as an antihistamine or an anti-inflammatory agent. .
  • the present invention provides a pharmaceutical composition comprising the antibody or fragment thereof according to the present invention.
  • the pharmaceutical composition may further include conventional pharmaceutically acceptable carriers and excipients.
  • the pharmaceutical composition may be applied to allergic diseases mediated by IgE. Representative diseases include, but are not limited to, allergic rhinitis, asthma, atopic dermatitis, urticaria and contact dermatitis.
  • the present invention is to advance the efficacy of a therapeutic agent that can be used for the diagnosis or treatment of allergic diseases, in particular allergic rhinitis, asthma, atopic skin disease, urticaria and contact dermatitis, comprising the antibody or fragment thereof according to the present invention.
  • a therapeutic agent that can be used for the diagnosis or treatment of allergic diseases, in particular allergic rhinitis, asthma, atopic skin disease, urticaria and contact dermatitis, comprising the antibody or fragment thereof according to the present invention.
  • compositions or kits for identifying and methods of diagnosing the disease or confirming therapeutic efficacy using such compositions or kits for identifying and methods of diagnosing the disease or confirming therapeutic efficacy using such compositions or kits.
  • the diagnosis of the disease or confirmation of the efficacy of the therapeutic agent may be performed by measuring the degree of inhibition of binding to IgE and Fc ⁇ RI in the blood of a patient using a rapid method using color development, but is not limited thereto.
  • 96-well plates and the like can also be used to quickly confirm the diagnostic or therapeutic efficacy of a large amount of samples.
  • the present invention also provides a polynucleotide sequence encoding the variable region of the antibody or fragment thereof according to the present invention.
  • polynucleotide sequences are polynucleotides set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and 39 May be selected from the sequence, and particularly preferably
  • the present invention provides a recombinant vector comprising the nucleotide sequence and a host cell comprising the same, and a method for producing an antibody that specifically binds to Fc ⁇ RI according to the present invention using the recombinant vector or host cell. Particularly, it is preferable to prepare by expression and purification by genetic recombination method. Specifically, the variable regions encoding the antibodies specifically binding to Fc ⁇ RI according to the present invention may be prepared separately or simultaneously by expression in one host cell. desirable.
  • a "recombinant vector” refers to a gene construct that is an expression vector capable of expressing a protein of interest in a suitable host cell, and which contains essential regulatory elements operably linked to express the gene insert.
  • "operably linked” means that the nucleic acid expression control sequence and the nucleic acid sequence encoding the protein of interest is functionally linked to perform a general function. Operative linkage with recombinant vectors can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation can be performed using enzymes generally known in the art. Can be easily used
  • Suitable expression vectors may include signal sequences for membrane targeting or secretion in addition to expression control elements such as promoters, initiation codons, termination codons, polyadenylation signals, and enhancers. Initiation and termination codons are generally considered to be part of the nucleotide sequence encoding the immunogenic target protein and must be functional in the subject and be in frame with the coding sequence when the gene construct is administered. Generic promoters can be either constitutive or inducible. Prokaryotic cells include, but are not limited to, lac, tac, T3 and T7 promoters.
  • Eukaryotic cells include monkey virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV), for example the long terminal repeat (LTR) promoter of HIV, moronivirus, cytomegalovirus (CMV) ), Epstein Barr virus (EBV), Loose sacoma virus (RSV) promoters, as well as promoters derived from ⁇ -actin promoter, human heroglobin, human muscle creatine, human metallothionein, but are not limited thereto.
  • the expression vector may comprise a selectable marker for selecting a host cell containing the vector.
  • the selection marker is for selecting cells transformed with the vector, and markers conferring a selectable phenotype such as drug resistance, nutritional requirements, resistance to cytotoxic agents or expression of surface proteins can be used. Since only cells expressing a selection marker survive in an environment treated with a selective agent, transformed cells can be selected.
  • the vector when the vector is a replicable expression vector, the vector may include a replication origin, which is a specific nucleic acid sequence from which replication is initiated.
  • various types of vectors such as plasmids, viruses, and cosmids can be used.
  • the type of recombinant vector is not particularly limited as long as it functions to express a desired gene and to produce a desired protein in various host cells of prokaryotic and eukaryotic cells, but has a promoter with strong activity and strong expression, similar to natural state. Vectors that can produce large amounts of foreign protein in form are preferred.
  • Suitable expression vectors for eukaryotic hosts may include, but are not limited to, expression control sequences derived from SV40, bovine papilloma virus, adenovirus, adeno-associated virus, cytomegalovirus and retrovirus. no.
  • Expression vectors that can be used in bacterial hosts include broader hosts such as bacterial plasmids obtained from Escherichia coli, such as pET, pRSET, pBluescript, pGEX2T, pUC vectors, col E1, pCR1, pBR322, pMB9, and derivatives thereof.
  • Plasmids with ranges, phage DNA that can be exemplified by a wide variety of phage lambda derivatives such as ⁇ gt10 and ⁇ gt11, NM989, and other DNA phages such as M13 and filamentary single-stranded DNA phages.
  • Useful expression vectors for yeast cells are 2 ° C. plasmids and derivatives thereof.
  • a useful vector for insect cells is pVL941.
  • the recombinant vector is inserted into a host cell to form a transformant.
  • Suitable host cells are Escherichia coli, Bacillus subtilis , Streptomyces sp., Pseudomonas sp., Proteus Prokaryotic cells such as Proteus mirabilis or Staphylococcus sp.
  • fungi such as Aspergillus sp., Pichia pastoris , Saccharomyces cerevisiae , Schizosaccharomyces sp.
  • Eukaryotic cells such as yeast, such as Spora crassa , other lower eukaryotic cells, and cells of higher eukaryotes, such as cells from insects.
  • the host cell is preferably derived from plants, mammals, monkey kidney cells (COS7) cells, NSO cells, SP2 / 0, Chinese hamster ovary (CHO: Chinese hamster ovary) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells and HEK293 cells and the like are available, but are not limited to these. Particularly preferably CHO cells.
  • Transformation into a host cell in the present invention includes any method of introducing a nucleic acid into an organism, cell, tissue or organ and can be carried out by selecting a suitable standard technique according to the host cell as is known in the art. These methods include electroporation, protoplast fusion, calcium phosphate (CaPO 4 ) precipitation, calcium chloride (CaCl 2 ) precipitation, agitation with silicon carbide fibers, agro bacterial mediated transformation, PEG, dextran sulfate, Lipofectamine and dry / inhibited mediated transformation methods and the like.
  • the medium and culture conditions can be appropriately selected and used according to the host cell. Conditions such as temperature, pH of the medium and incubation time can be appropriately adjusted to be suitable for the growth of cells and the mass production of proteins during the culture.
  • the recombinantly produced antibody or antibody fragment as described above can be recovered from the medium or cell digest, and can be isolated and purified by conventional biochemical separation techniques (Sambrook et al., Molecular Cloning: A laborarory Manual, 2nd).
  • Antibodies that specifically bind to Fc ⁇ RI according to the present invention efficiently inhibit the binding between IgE and Fc ⁇ RI at low concentrations, such as allergic rhinitis, asthma, atopic skin disease, urticaria and contact dermatitis mediated by IgE. It shows excellent therapeutic effect on various allergic diseases, and because it specifically binds and works only on Fc ⁇ RI, there are almost no side effects and does not cause an immune response when administered in the body.
  • Fc ⁇ RI specific antibodies according to the present invention may inhibit or treat IgE mediated allergic diseases by inhibiting binding between IgE and Fc ⁇ RI.
  • IgE mediated allergic diseases such as allergic rhinitis, asthma, atopic skin disease, urticaria and contact dermatitis.
  • 1 is a diagram showing the inhibitory effect of Fc ⁇ RI and IgE on selected antibodies using a competitive ELISA technique.
  • Figure 3 shows the degranulation inhibitory effect of selected antibodies.
  • FIG. 5 shows changes in total cell number, macrophages, eosinophils and lymphocytes by selected antibodies in an OVA induced Fc ⁇ RI ⁇ transgenic mouse asthma model.
  • Figure 6 shows the size exclusion chromatography results of the Fc ⁇ RI specific antibody NPB311 according to the present invention.
  • Figure 7 confirms the structural stability of the Fc ⁇ RI specific antibody NPB311 according to the present invention.
  • Figure 8 shows the degranulation inhibitory effect of the Fc ⁇ RI specific antibody NPB311 according to the present invention.
  • FIG. 9 is a view showing the effect of Fc ⁇ RI specific antibody NPB311 according to the present invention on the secretion of beta-hexos aminidayes.
  • FIG. 10 is a view showing the histamine secretion inhibitory effect of the Fc ⁇ RI specific antibody NPB311 according to the present invention.
  • FIG. 11 is a view showing the effect of Fc ⁇ RI specific antibody NPB311 according to the present invention on the induction of histamine secretion.
  • FIG. 12 is a diagram showing the hypersensitivity inhibitory effect of Fc ⁇ RI specific antibody NPB311 according to the present invention.
  • scFv DNA having randomly bound immunoglobulin heavy (H chain) and light (L chain) cDNAs was prepared, and a scFv phage library consisting of 10 10 clones was prepared by introducing a phagemid vector.
  • the phage library is composed of two different libraries: a phage library made by synthesizing the CDR sequences of various human immunoglobulins in the form of a single chain scFv, and a phage library of scFv made of cDNA extracted from human peripheral blood. It includes.
  • the phage clones are then screened for display of scFv having the property of binding to human Fc ⁇ RI ⁇ via a panning method, which is brought into contact with the soluble fragment of the ⁇ subunit (Fc ⁇ RI ⁇ ) of human Fc ⁇ RI immobilized on a solid phase. , Obtained.
  • Fc ⁇ RI was coated on an Immuno tube at an concentration of 10 ug / ml at 4 ° C. for at least 12 hours.
  • a phage library expressing scFv of human antibody was added to the tube and allowed to react for 1 hour, followed by 0.01% Tween-20. Washed 10 times with PBS (phosphate buffered saline) and phage eluted with 0.1M triethylamine. Phage ELISA (ELISA) was then performed to select phages with strong reactivity to Fc ⁇ RI and to inhibit the interaction of Fc ⁇ RI and IgE.
  • 96-well plates are coated with 0.5 ⁇ g / ml Fc ⁇ RI and then stopped with 2% skim milk.
  • the Fc ⁇ RI specific antibodies selected in Example 1 are diluted and added to each well and reacted at 37 ° C. for 1 hour.
  • PBS / Tween solution After washing the 96-well plate with PBS / Tween solution, 0.5 ug / ml of human IgE was added to each well and reacted at 37 ° C. for 1 hour, followed by washing with PBS / Twin again and then anti-IgE -HRP (1 ug / ml) is added to react for 1 hour at 37 °C. Thereafter, 30 ⁇ l of the luminous reagent was added to measure light emission.
  • the selected antibodies showed a concentration-dependent effect of inhibiting the binding of Fc ⁇ RI and IgE (see Fig. 1).
  • the selected antibodies NPB 302, NPB 303, NPB 305, NPB 307 and NPB 311 all have a binding ability to human Fc ⁇ RI (see Fig. 2).
  • RBL-SX38 cells were suspended in 3% FBS and DMEM medium in 96-well plates for beta hexosminidayes secretion, and then 37 ° C., 5% CO 2 at 2 ⁇ 10 4 cells / 100 ul in 96-plate. Incubated for 48 hours in the incubator.
  • each cell was washed once with PBS + a buffer (PBS, 0.4 mM MgCl 2 , 1 mM CaCl 2 , 5.6 mM glucose, 0.1% BSA), followed by nitropheny-IgE (NP-).
  • IgE) 0.1 ⁇ g / ml was dispensed and then reacted for 30 minutes in a 37 ° C., 5% CO 2 incubator, followed by diluting the selected Fc ⁇ RI specific antibody to each well and reacting for 150 minutes in a 37 ° C., 5% CO 2 incubator. I was.
  • OVA ovalbuproliferin
  • Alum 100 ug of OVA (ovalbumin) and 2 mg of Alum were included in 200 ⁇ l solution.
  • the mixed OVA-alum was continuously mixed at 37 ° C. for about 1 hour.
  • the mixed OVA-alum was divided into 200 ⁇ l in a 1.5 ml tube and placed one by one in a 1 ml syringe. Subsequently, 6-week-old females transformed to express human Fc ⁇ RI were intraperitoneally injected 200 ⁇ l over 0, 14 days and 2 times. On 28, 29 and 30 days, 30 ⁇ g of 1% OVA was inhaled through the nasal mucosa.
  • Selected antibodies NPB 302 and NPB 311 were injected at 27, 29 days at 3 mg / kg. After 24 hours the cell number in the bronchoalveolar lavage fluid was counted.
  • the bronchial tubes of anesthetized mice were incised using zoletil, a tube was inserted, a 22-gauge needle was inserted into the trachea, and bronchoalveolar lavage was performed.
  • the samples were collected by injecting 0.9 mL of PBS twice per time. .
  • the collected bronchial alveolar lavage fluid was centrifuged at 4 ° C. and 5000 rpm for 5 minutes, and the supernatant was stored at ⁇ 70 ° C. until the next experiment.
  • Cells obtained by centrifugation were counted using a hemocytometer and plated on glass slides using cytospin 3 (Thermo, USA), and Diff-Quik ) Staining was performed. A total of 200 cells were counted to differentially calculate alveolar macrophages, eosinophils, lymphocytes and neutrophils.
  • Dialysis membrane (Membra-Cel, Width 25mm, Diameter 16mm, MWCO: 3500) in 10 mM Ammonium carbonate buffer (pH 7.2) was dialyzed at 4 ° C. overnight. After storage at ⁇ 80 ° C., Trep Tem. Mot. Pin31 (centrifuge for Vacuum concentrator) was used. : -88 °C, Vacuum Pump: lyophilized at 9 Torr conditions.
  • RBL-SX38 cells were suspended in 3% FBS (Feral Bovine Serum) and DMEM medium (Dulbecco's Modified Eagle's Medium) in 96-well plates for determination of beta hexosminidayes secretion, followed by 2 ⁇ 10 4 in 96-plate. The cells / 100 ul were incubated for 48 hours in a 37 °C, 5% CO 2 incubator.
  • each cell was washed once with PBS + a buffer (PBS, 0.4 mM MgCl 2 , 1 mM CaCl 2 , 5.6 mM glucose, 0.1% BSA) and then nitropheny-IgE (NP-; IgE) 0.1 ⁇ g / ml was dispensed and reacted for 30 minutes in a 37 ° C., 5% CO 2 incubator.
  • PBS 0.4 mM MgCl 2 , 1 mM CaCl 2 , 5.6 mM glucose, 0.1% BSA
  • NP-; IgE nitropheny-IgE
  • NPB-311 (Fab) was treated for each concentration and reacted for 150 minutes in 37 °C, 5% CO 2 incubator. After adding NP-BSA (0.1 ug / ml) for 30 minutes at 37 °C, 5% CO 2 incubator and 30 ⁇ l of the supernatant containing beta hexosminidayes secreted out of the cell 0.1% Triton X-100 Lysis with 60 ⁇ l yielded beta hexosminiminides in cells.
  • RBL-SX38 cells were suspended in 3% FBS (Feral Bovine Serum) and DMEM medium (Dulbecco's Modified Eagle's Medium) in 96-well plates for measuring beta hexosminidayes secretion, and then 2 ⁇ 10 4 cells in 96-plate Incubated for 48 hours at 37 °C, 5% CO 2 incubator at / 100 ul.
  • FBS Feal Bovine Serum
  • DMEM medium Dulbecco's Modified Eagle's Medium
  • each cell was washed once with PBS + a buffer (PBS, 0.4 mM MgCl 2 , 1 mM CaCl 2 , 5.6 mM glucose, 0.1% BSA), and then 15 ug / ml NPB-311 (Fab) was added.
  • PBS + a buffer PBS, 0.4 mM MgCl 2 , 1 mM CaCl 2 , 5.6 mM glucose, 0.1% BSA
  • 15 ug / ml NPB-311 (Fab) was added.
  • the cells were added and reacted sequentially for 0, 2, 5, 10, 30, and 60 minutes, and NP-BSA (0.1 ug / ml) was added thereto for 30 minutes in a 37 ° C., 5% CO 2 incubator.
  • RBL-SX38 cells were suspended in 10-well FBS and DMEM medium in 24-well plates, then 37 ° C., 5% CO 2 at 2 ⁇ 10 5 cells / 500 ul in 24-well plates. Incubated for 48 hours in the incubator.
  • each cell was loaded with Tyrode's buffer (125 mM NaCl, 5 mM KCl, 0.4 mM MgCl 2 , 1 mM CaCl 2 , 5.6 mM glucose, 0.1% BSA, 10 mM Hepes, pH7. After washing twice with 2), 0.5 ⁇ g / ml of NP-IgE was dispensed and reacted in a culture medium at 37 ° C. in a 5% CO 2 incubator for 24 hours. At this time, NPB-311 (Fab) was treated together. After 24 hours, the reaction was washed three times with T-load buffer, and then 0.1 ug / ml NP-BSA was added thereto, followed by reaction for 30 minutes in a 37 ° C, 5% CO 2 incubator.
  • Tyrode's buffer 125 mM NaCl, 5 mM KCl, 0.4 mM MgCl 2 , 1 mM CaCl 2 , 5.6 m
  • RBL-SX38 cells were first suspended in 10-well FBS and DMEM medium in 24-well plates, followed by 37 ° C. and 5% CO 2 incubator at 2 ⁇ 10 5 cells / 500 ul in 24-well plates. Incubated for 48 hours.
  • each cell was loaded with Tyrode's buffer (125 mM NaCl, 5 mM KCl, 0.4 mM MgCl 2 , 1 mM CaCl 2 , 5.6 mM glucose, 0.1% BSA, 10 mM Hepes, pH7). .2) twice and then NPB-311 (Fab) 15 ug / ml was added to the cells and reacted sequentially for 0, 2, 5, 10, 30, 60 minutes and then NP-BSA (0.1 ug / ml ) Was added and reacted at 37 ° C. in a 5% CO 2 incubator for 30 minutes.
  • Tyrode's buffer 125 mM NaCl, 5 mM KCl, 0.4 mM MgCl 2 , 1 mM CaCl 2 , 5.6 mM glucose, 0.1% BSA, 10 mM Hepes, pH7. .2
  • NPB-311 (Fab) 15 ug / ml was added to the cells and
  • the supernatant of the reaction was centrifuged to obtain a supernatant, 100 ⁇ l of the obtained supernatant was transferred to a 96-well plate and acetylated histamine, and then the histamine content was determined using an HTRF Kit (CISBIO International, France). Measured.
  • NPB311 (Fab) was diluted 1/2 to HBS-EP buffer with 100 to 0 nM 6point and flowed to the CM5 chip in the order of low to high concentration to measure kinetics and regenerated using 10 mM NaOH buffer.
  • the affinity between NPB311 (Fab) and sFc ⁇ RI was measured by Biacore (SPR).
  • KD 4.07 ⁇ 10 -9 (M) was measured to determine that the affinity of NPB311 (Fab) and sFc ⁇ RI is good.
  • RBL-SX38 cells were added to 5 x 10E4 / 100 ul / well in black wall 96 wells, and after 37 ° C overnight incubation, NP-IgE to final 100 ng / ml or NPB-311 (Fab) to NP-IgE 500 , 100, 20, 4, 0.8, 0.16, 0 nM was added to 37 °C, incubation for 2 hours. After washing once with PBS, FLIPR calcium 4 dye was added thereto, and incubation at 37 ° C. for 1 hour was followed by NP-BSA. The fluoresence of FLUO-4AM was measured 90 times at 2 second intervals.
  • NPB-311 (Fab) was found to inhibit the release of calcium (calcium) in a concentration-dependent manner, From the above results, it was confirmed that the NPB-311 (Fab) antibody portion was coupled to Fc ⁇ RIa to inhibit dimerization.
  • mice female 7-week-old mice were injected subcutaneously with 0.1 ug NP-IgE 20 ul in the left ear on Day 0, while simultaneously adding 0.1 ug NP-IgE 20 ul and 0.05 MPK NPB-311 (Fab) antibody Subcutaneous injection into the ear. However, at this time, the antibody was divided into two groups, which were measured 30 minutes after the antibody injection and the next day, which were measured.
  • Fab MPK NPB-311
  • NPB-311 Fab
  • human Fc ⁇ RIa expressing transgenic mice it was confirmed that anaphylaxis was suppressed. From the above results, NPB-311 (Fab) was bound to IgE-Fc ⁇ RIa. It was confirmed that it interferes with (see Figure 12).
  • SEQ ID NO: 1 shows the nucleotide sequence of the heavy chain variable region HC1
  • SEQ ID NO: 2 shows the amino acid sequence of the heavy chain variable region HC1
  • SEQ ID NO: 3 shows the nucleotide sequence of the heavy chain variable region HC2
  • SEQ ID NO: 4 shows the amino acid sequence of the heavy chain variable region HC2
  • SEQ ID NO: 5 shows the nucleotide sequence of the heavy chain variable region HC3
  • SEQ ID NO: 6 shows amino acid sequence of heavy chain variable region HC3
  • SEQ ID NO: 7 shows the nucleotide sequence of the heavy chain variable region HC4
  • SEQ ID NO: 8 shows the amino acid sequence of the heavy chain variable region HC4
  • SEQ ID NO: 9 shows the nucleotide sequence of the heavy chain variable region HC5
  • SEQ ID NO: 10 shows the amino acid sequence of the heavy chain variable region HC5
  • SEQ ID NO: 11 shows the nucleotide sequence of the heavy chain variable region HC6
  • SEQ ID NO: 12 shows the amino acid sequence of the heavy chain variable region HC6
  • SEQ ID NO: 13 shows the nucleotide sequence of the heavy chain variable region HC7
  • SEQ ID NO: 14 shows amino acid sequence of heavy chain variable region HC7
  • SEQ ID NO: 15 shows the nucleotide sequence of the heavy chain variable region HC8
  • SEQ ID NO: 16 shows amino acid sequence of heavy chain variable region HC8
  • SEQ ID NO: 17 shows the nucleotide sequence of the light chain variable region LC1;
  • SEQ ID NO: 18 shows amino acid sequence of light chain variable region LC1
  • SEQ ID NO: 19 shows the nucleotide sequence of the light chain variable region LC2
  • SEQ ID NO: 20 shows amino acid sequence of light chain variable region LC2
  • SEQ ID NO: 21 shows the nucleotide sequence of the light chain variable region LC3
  • SEQ ID NO: 22 shows the amino acid sequence of the light chain variable region LC3
  • SEQ ID NO: 23 shows the nucleotide sequence of the light chain variable region LC4
  • SEQ ID NO: 24 shows the amino acid sequence of the light chain variable region LC4
  • SEQ ID NO: 25 shows the nucleotide sequence of the light chain variable region LC5
  • SEQ ID NO: 26 shows the amino acid sequence of the light chain variable region LC5
  • SEQ ID NO: 27 shows the nucleotide sequence of the light chain variable region LC6
  • SEQ ID NO: 28 shows amino acid sequence of light chain variable region LC6
  • SEQ ID NO: 29 shows the nucleotide sequence of the light chain variable region LC7
  • SEQ ID NO: 30 shows amino acid sequence of light chain variable region LC7
  • SEQ ID NO: 31 shows the nucleotide sequence of the light chain variable region LC8
  • SEQ ID NO: 32 shows amino acid sequence of light chain variable region LC8
  • SEQ ID NO: 33 shows the nucleotide sequence of the light chain variable region LC9
  • SEQ ID NO: 34 shows amino acid sequence of light chain variable region LC9
  • SEQ ID NO: 35 shows the nucleotide sequence of the light chain variable region LC10
  • SEQ ID NO: 36 shows amino acid sequence of light chain variable region LC10
  • SEQ ID NO: 37 shows the nucleotide sequence of the light chain variable region LC11
  • SEQ ID NO: 38 shows the amino acid sequence of the light chain variable region LC11
  • SEQ ID NO: 39 shows the nucleotide sequence of the light chain variable region LC12
  • SEQ ID NO: 40 shows the amino acid sequence of the light chain variable region LC12.

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Abstract

La présente invention concerne un anticorps humain spécifique de FcεRI, qui est un récepteur possédant une affinité élevée pour le Fc de l'IgE, et concerne également un procédé pour le préparer. L'anticorps humain spécifique de FcεRI selon la présente invention inhibe efficacement le couplage entre l'IgE et FcεRI, et peut être utilisé pour traiter ou diagnostiquer des maladies allergiques induites par l'IgE, comme la rhinite allergique, l'asthme, la dermatite atopique, l'urticaire et la dermatite contagieuse.
PCT/KR2012/004247 2011-06-07 2012-05-30 Anticorps humain spécifique de fcεri et composition le contenant pour traiter ou diagnostiquer des maladies allergiques WO2012169741A2 (fr)

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KR1020120057081A KR20120135868A (ko) 2011-06-07 2012-05-30 ＦｃεＲＩ 특이적 인간 항체 및 이를 포함하는 알레르기 질환 치료 또는 진단용 조성물

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018119413A1 (fr) * 2016-12-22 2018-06-28 Lipidair, Llc Procédés et compositions d'administration ciblée pour des antihistaminiques

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Publication number Priority date Publication date Assignee Title
JPH11174A (ja) * 1997-06-13 1999-01-06 Asahi Breweries Ltd 酵母を用いた抗体Ｆａｂ断片の製造法
WO2002102320A2 (fr) * 2001-06-15 2002-12-27 Tanox, Inc. PROTEINES DE FUSION FCε POUR LE TRAITEMENT DE L'ALLERGIE ET DE L'ASTHME
US20070135621A1 (en) * 2003-04-03 2007-06-14 Laboratoire Francais Du Fractionnement Et Des Biotechnologies Therapeutic products with enhance ability to immunomodulate cell functions
KR100932465B1 (ko) * 2001-07-19 2009-12-17 자이단호진 가가쿠오요비겟세이료호겐쿠쇼 인간형 항인간 ＩｇＥ 수용체 항체 및 항체 단편
WO2012037196A1 (fr) * 2010-09-14 2012-03-22 The Henry M. Jackson Foundation Procédés de traitement de maladies auto-immunes avec des anticorps anti-fceri

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Publication number Priority date Publication date Assignee Title
JPH11174A (ja) * 1997-06-13 1999-01-06 Asahi Breweries Ltd 酵母を用いた抗体Ｆａｂ断片の製造法
WO2002102320A2 (fr) * 2001-06-15 2002-12-27 Tanox, Inc. PROTEINES DE FUSION FCε POUR LE TRAITEMENT DE L'ALLERGIE ET DE L'ASTHME
KR100932465B1 (ko) * 2001-07-19 2009-12-17 자이단호진 가가쿠오요비겟세이료호겐쿠쇼 인간형 항인간 ＩｇＥ 수용체 항체 및 항체 단편
US20070135621A1 (en) * 2003-04-03 2007-06-14 Laboratoire Francais Du Fractionnement Et Des Biotechnologies Therapeutic products with enhance ability to immunomodulate cell functions
WO2012037196A1 (fr) * 2010-09-14 2012-03-22 The Henry M. Jackson Foundation Procédés de traitement de maladies auto-immunes avec des anticorps anti-fceri

Non-Patent Citations (1)

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Title
SHUHEI HASHIGUCHI ET AL.: 'Human FceRla-specific human single-chain Fv (scFv) antibody with antagonistic activity toward IgE/FceRIa-binding' THE JOURNAL OF BIOCHEMISTRY vol. 133, no. 1, 2003, ISSN 0021-924X pages 43 - 49 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018119413A1 (fr) * 2016-12-22 2018-06-28 Lipidair, Llc Procédés et compositions d'administration ciblée pour des antihistaminiques
US10954307B2 (en) 2016-12-22 2021-03-23 Lipidair, Llc Targeted delivery methods and compositions for antihistamines

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