US20180312566A1 - Fc FUSION HIGH AFFINITY IgE RECEPTOR ALPHA-CHAIN - Google Patents

Fc FUSION HIGH AFFINITY IgE RECEPTOR ALPHA-CHAIN Download PDF

Info

Publication number
US20180312566A1
US20180312566A1 US16/034,648 US201816034648A US2018312566A1 US 20180312566 A1 US20180312566 A1 US 20180312566A1 US 201816034648 A US201816034648 A US 201816034648A US 2018312566 A1 US2018312566 A1 US 2018312566A1
Authority
US
United States
Prior art keywords
protein
present
amino acid
acid sequence
high affinity
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US16/034,648
Inventor
Takashi Sakamoto
Yoichi Inada
Kazumasa Yokoyama
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Kissei Pharmaceutical Co Ltd
Original Assignee
Kissei Pharmaceutical Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kissei Pharmaceutical Co Ltd filed Critical Kissei Pharmaceutical Co Ltd
Priority to US16/034,648 priority Critical patent/US20180312566A1/en
Publication of US20180312566A1 publication Critical patent/US20180312566A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70535—Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention relates to an Fc fusion high affinity IgE receptor ⁇ -chain that is useful as a pharmaceutical product.
  • the present invention relates to an Fc fusion high affinity IgE receptor ⁇ -chain having excellent stability at low pH, and a medicinal use thereof.
  • Immunoglobulin E is one of an immunoglobulin group, which plays a role in allergic reactions.
  • the IgE which is secreted from B cells or is expressed on the surface of the B cells, binds to a high affinity IgE receptor (Fc ⁇ RI) found on the surface of mast cells, basophils, etc.
  • Fc ⁇ RI high affinity IgE receptor
  • the IgE becomes a form in which it crosslinks the antigen.
  • chemical mediators such as histamine or serotonin, which are stored in intracellular granules, are released. Consequently, an inflammatory reaction is induced, and type I allergy symptoms such as telangiectasis or vascular hyperpermeability are provoked (Non Patent Literature 1).
  • a compound or a protein which inhibits the binding of IgE to Fc ⁇ RI, inhibits the binding of the IgE to the Fc ⁇ RI found on the surface of mast cells, basophils, etc.
  • a compound or a protein is expected as a therapeutic agent for type I allergic diseases such as bronchial asthma, allergic rhinitis, and allergic conjunctivitis (Non Patent Literature 2).
  • Etanercept has been known as a therapeutic agent for rheumatoid arthritis.
  • Etanercept is a completely humanized soluble TNF ⁇ /LT ⁇ receptor formulation, which has been focused because of the role of a soluble receptor of a tumor necrosis factor (TNF) to suppress the action of TNF in a living body, and has been then developed.
  • TNF tumor necrosis factor
  • the protein pharmaceutical product can be expected to have high therapeutic effects. On the other hand, it may cause a problem specific to the protein pharmaceutical product in the production process thereof.
  • a purification method of using protein A is applied.
  • a buffer with a low pH value is used to elute a protein of interest that binds to the protein A.
  • the protein of interest is desirably treated at low pH for a certain period of time.
  • a protein which is poor in stability at low pH, easily forms aggregates. If the ratio of aggregates is high, a reduction in purification efficiency or production amount occurs in the production of protein pharmaceutical products. In addition, an immune response is provoked by mixing aggregates into pharmaceutical products, and as a result, serious side effects, such as anaphylaxis, are likely to occur.
  • the instability of a protein of interest at low pH may be problematic in the production of protein pharmaceutical products.
  • Patent Literature 1 discloses a high affinity IgE receptor as an example of such immunoadheson. However, this publication does not specifically describe a fused protein of a high affinity IgE receptor and an immunoglobulin.
  • Fusion protein A A fused protein (hereinafter referred to as “Fusion protein A”) of a high affinity IgE receptor ⁇ -chain (Fc ⁇ RI ⁇ -chain; hereinafter referred to as “FCER1A”) and immunoglobulin G1 (IgG1) is disclosed in Non Patent Literature 3.
  • FCER1A high affinity IgE receptor ⁇ -chain
  • IgG1 immunoglobulin G1
  • the Fusion protein A disclosed in the aforementioned publication is largely different from the protein of the invention of the present application, in terms of the mode of binding FCER1A to IgG1 (Fc). That is to say, the protein of the invention of the present application has a characteristic amino acid sequence in a linker fragment region between the Fc ⁇ RI and the IgG1.
  • a fused protein (NPB301) formed by linking a water-soluble fragment of the high affinity IgE receptor (Fc ⁇ RI) to a human Fc region via a peptide linker is disclosed in Patent Literature 2.
  • the protein of the invention of the present application does not comprise the peptide linker disclosed in Patent Literature 2.
  • Patent Literature 2 neither discloses nor suggests the characteristic amino acid sequence of the linker fragment region of the present invention.
  • a fused protein of FCER1A and immunoglobulin G2 (IgG2) is disclosed in Patent Literature 3.
  • this fused protein comprising IgG2 is different from the protein of the invention of the present application, in terms of the amino acid sequences of a linker fragment region and an Fc region.
  • Patent Literature 3 and Patent Literatures 1 to 7 neither disclose nor suggest the protein of the present invention.
  • the present inventors have conducted intensive studies in order to obtain an Fc fusion high affinity IgE receptor ⁇ -chain having high stability against low pH or heat. As a result, the inventors have found that an Fc fusion high affinity IgE receptor ⁇ -chain having high stability can be obtained by using a linker fragment comprising three Cys residues, in a fused protein comprising a high affinity IgE receptor ⁇ -chain and the Fc region of IgG1, thereby completing the present invention.
  • the present invention is as follows.
  • the present invention relates to the following [1] to [5], etc.
  • a linker fragment region between the (i) and the (ii) is the amino acid sequence shown in SEQ ID NO: 2.
  • the present description includes the contents as disclosed in Japanese Patent Application No. 2015-032231 and 2015-252231, which are priority documents of the present application.
  • the protein of the present invention has excellent stability at low pH.
  • the protein of the present invention has excellent neutralizing activity against IgE. Accordingly, the protein of the present invention is useful as a protein pharmaceutical product for preventing or treating type I allergic diseases mediated by IgE.
  • FIG. 1 shows the activity of inhibiting the binding of human IgE.
  • the horizontal axis indicates the concentration of each drug (mol/L); and the longitudinal axis indicates the value of the amount of IgE bound to Protein 1 immobilized on a plate, which is shown at a percentage (free IgE (percent with respect to a control)), using, as a reference, the binding amount obtained when a predetermined amount of IgE is added.
  • the circle indicates the value of Protein 1
  • the square indicates the value of Omalizumab, respectively.
  • FIG. 2 shows a transition of a change in the content rate (%) of aggregates at low pH.
  • the horizontal axis indicates the number of days (Day), and the longitudinal axis indicates a change in the content rate (%) of aggregates.
  • the circle indicates the value of Protein 1
  • the square indicates the value of Fusion protein A, respectively.
  • FIG. 3 shows a transition of a change in the content rate (%) of aggregates by a heat treatment.
  • the horizontal axis indicates the number of days (Day), and the longitudinal axis indicates a change in the content rate (%) of aggregates.
  • the circle indicates the value of Protein 1
  • the square indicates the value of Fusion protein A, respectively.
  • the “high affinity IgE receptor ⁇ -chain (FCER1A)” means a protein comprising an ⁇ -chain portion that is an extracellular domain of a high affinity IgE receptor.
  • the high affinity IgE receptor ⁇ -chain is, for example, a protein shown in the following SEQ ID NO: 1.
  • SEQ ID NO: 1 VPQKPKVSLNPPWNRIFKGENVTLTCNGNNFFEVSSTKWFHNGSLSEETN SSLNIVNAKFEDSGEYKCQHQQVNESEPVYLEVFSDWLLLQASAEVVMEG QPLFLRCHGWRNWDVYKVIYYKDGEALKYWYENHNISITNATVEDSGTYY CTGKVWQLDYESEPLNITVIKAPREKYWL
  • the above-described high affinity IgE receptor ⁇ -chain includes, for example, a protein having identity of 90% or more, 95% or more, 97% or more, or 99% or more to the amino acid sequence shown in SEQ ID NO: 1, when the identity is calculated using BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) or the like (e.g., using default parameters), and having binding ability to IgE.
  • BLAST Basic Local Alignment Search Tool at the National Center for Biological Information
  • the high affinity IgE receptor ⁇ -chain also includes a protein comprising an amino acid sequence having a substitution, deletion and/or addition of one or more, or several amino acids (1 to 10, preferably 1 to 5, and more preferably 1 or 2 amino acids) with respect to the amino acid sequence shown in SEQ ID NO: 1, and having binding ability to IgE.
  • the “Fc region of IgG1” means the Fc fragment of immunoglobulin G1, namely, the CH2 and CH3 constant domains of native immunoglobulin G1.
  • the Fc region of IgG1 includes all of a natural mutant, an artificial mutant, and a truncated form.
  • the “linker fragment region between the high affinity IgE receptor ⁇ -chain and the Fc region of IgG1” means a region consisting of 14 amino acid residues ranging from the junction point between the above-described high affinity IgE receptor ⁇ -chain and the above described Fc region of IgG1 towards the direction of the Fc region.
  • the “Fc fusion protein” means a recombinant protein comprising the high affinity IgE receptor ⁇ -chain and the Fc fragment of an immunoglobulin.
  • the protein of the present invention is characterized in that the linker fragment region between the high affinity IgE receptor ⁇ -chain and the Fc region of IgG1 is the amino acid sequence shown in the following SEQ ID NO: 2.
  • SEQ ID NO: 2 EPKSCDKTHTCPPC
  • the protein of the present invention is preferably an Fc fusion protein comprising the amino acid sequence shown in the following SEQ ID NO: 3 (hereinafter referred to as “Protein 1”).
  • SEQ ID NO: 3 VPQKPKVSLNPPWNRIFKGENVTLTCNGNNFFEVSSTKWFHNGSLSEETN SSLNIVNAKFEDSGEYKCQHQQVNESEPVYLEVFSDWLLLQASAEVVMEG QPLFLRCHGWRNWDVYKVIYYKDGEALKYWYENHNISITNATVEDSGTYY CTGKVWQLDYESEPLNITVIKAPREKYWLEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFScSVMHEALHNHY T
  • the amino acid sequence shown in SEQ ID NO: 3 has a sequence formed by fusing the amino acid sequence shown in SEQ ID NO: 1 (from Val at position 1 to Leu at position 179 of the amino acid sequence shown in SEQ ID NO: 3), the amino acid sequence shown in SEQ ID NO: 2 (Glu at position 180 to Cys at position 193 of the amino acid sequence shown in SEQ ID NO: 3), and the amino acid sequence of the Fc fragment of an immunoglobulin (Pro at position 194 to Lys 411 of the amino acid sequence shown in SEQ ID NO: 3) in this order.
  • This Fc fusion protein includes a protein comprising an amino acid sequence having identity of 90% or more, 95% or more, 97% or more, or 99% or more to the amino acid sequence shown in SEQ ID NO: 3, which is other than the amino acid sequence portion from Glu at position 180 to Cys at position 193 that corresponds to the amino acid sequence shown in SEQ ID NO: 2, when the identity is calculated using, for example, BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) or the like (e.g., using default parameters), and having binding ability to IgE.
  • BLAST Basic Local Alignment Search Tool at the National Center for Biological Information
  • this Fc fusion protein includes a protein comprising an amino acid sequence having a substitution, deletion and/or addition of one or more, or several amino acids (1 to 10, preferably 1 to 5, and more preferably 1 or 2 amino acids) with respect to the amino acid sequence shown in SEQ ID NO: 3, which is other than the amino acid sequence portion from Glu at position 180 to Cys at position 193 that corresponds to the amino acid sequence shown in SEQ ID NO: 2, and having binding ability to IgE.
  • the protein of the present invention may be an Fc fusion protein comprising an amino acid sequence having a deletion of the lysine (K) at the C-terminus of the above-described Protein 1.
  • an Fc fusion protein comprising an amino acid sequence having a deletion of the lysine (K) at the C-terminus of Protein 1 shown in SEQ ID NO: 3 consists of an amino acid sequence portion from the position 1 to the position 410 of the amino acid sequence shown in SEQ ID NO: 3.
  • the protein of the present invention includes both a monomer and a dimer of an Fc fusion protein formed by linking a high affinity IgE receptor ⁇ -chain to the Fc region of IgG1 via a linker fragment consisting of the amino acid sequence shown in SEQ ID NO: 2.
  • a linker fragment consisting of the amino acid sequence shown in SEQ ID NO: 2.
  • Three Cys residues are present in the above-described linker fragment region (the Cys residues at positions 184, 190, and 193 of the amino acid sequence shown in SEQ ID NO: 3), and a dimer can be formed by disulfide bonds.
  • two Fc fusion protein monomers form a dimer as a result of the formation of three disulfide bonds between Cys residues at the same positions as those of the above-described three Cys residues, and the above-described three Cys residues. Because of the above-described three disulfide bonds, a dimer is stabilized, and it has high stability against low pH and heat.
  • the phrase “having high stability against low pH and heat” means, for example, that only a few aggregates are formed under conditions of low pH and under heating. Such high stability against low pH or heat can be confirmed, for example, by performing a low pH treatment or a heat treatment on a protein, and then measuring the content of aggregates in the protein by gel filtration chromatography.
  • the Fc fusion protein of the present invention is preserved at a low temperature of 2° C. to 8° C., and preferably 4° C., at pH 1 to 5, and preferably at pH 2 to 4, for 1 day to 1 month, preferably for 1 day to 14 days, and more preferably for 5 to 12 days, or even if the present Fc fusion protein is preserved at a temperature of 25° C. to 45° C., and preferably 30° C. to 40° C., for 1 day to 1 month, preferably 1 day to 14 days, and more preferably 1 day to 7 days, a change in the content rate of aggregates is small.
  • a change in the content rate of aggregates in the protein of the present invention is calculated based on the peak area of gel filtration chromatography, it is 10% or less, and preferably 8% or less. Further, when compared with an Fc fusion protein having two or less Cys residues as a linker fragment, a change in the content rate of aggregates in the protein of the present invention is small.
  • the protein of the present invention can be produced, for example, by the following method or a method equivalent thereto, or by the methods described in publications or methods equivalent thereto.
  • the protein of the present invention can be produced using a genetic recombination technique that is well known to a person skilled in the present technical field. For example, DNA encoding the protein of the present invention is prepared, and an expression vector comprising this DNA is then constructed. Subsequently, prokaryotic or eukaryotic cells are transformed or transfected with the above-described vector, and a protein of interest can be isolated and purified from a culture supernatant of the obtained cells.
  • the protein of the present invention can also be produced using protein-expressing cells that are well known to a person skilled in the present technical field.
  • cDNA encoding the amino acid sequence shown in SEQ ID NO: 3 is incorporated into a mammalian expression plasmid vector to prepare a protein expression plasmid, and the prepared plasmid is then introduced into animal cells such as Chinese hamster ovary cells (CHO), so as to establish a stable expression cell line.
  • animal cells such as Chinese hamster ovary cells (CHO), so as to establish a stable expression cell line.
  • the obtained cells are cultured, and then, the protein of the present invention can be obtained from a culture supernatant.
  • the protein of the present invention can be isolated and purified, as necessary, by isolation and purification means that are well known to a person skilled in the present technical field.
  • isolation and purification means include affinity chromatography, ion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, mixed mode chromatography, dialysis, a fractional precipitation method, and electrophoresis.
  • the protein of the present invention can also be isolated and purified by combining these methods with one another, as appropriate.
  • the protein of the present invention may also undergo chemical modification that is well known to a person skilled in the present technical field.
  • chemical modification include glycosylation, polyethylene glycolation (PEG), acetylation, and amidation.
  • the protein of the present invention Since the protein of the present invention has neutralizing activity against IgE, it can be used as a preventive or therapeutic agent for various diseases mediated by IgE.
  • the protein of the invention of the present application is useful as a preventive or therapeutic agent for diseases associated with type I allergy, and the like, such as bronchial asthma, eosinophilic otitis media, eosinophilic sinusitis, allergic conjunctivitis, allergic rhinitis, pollinosis, food allergy, mite allergic disease, hives, and anaphylactic shock.
  • the protein of the present invention has excellent affinity for IgE.
  • the protein of the present invention can also be used as a “protein-drug conjugate” that utilizes such affinity, such as an antibody-drug conjugate (ADC).
  • ADC antibody-drug conjugate
  • Examples of such a “protein-drug conjugate” include the modes of use, such as “Protein 1-drug” and “Protein 1-linker-drug.”
  • As a drug an anti-allergic agent or the like can be used.
  • Such a conjugate can be produced by a method well known to a person skilled in the present technical field.
  • an oral agent examples include a tablet, a powder agent, a granule, a fine granule, and a capsule.
  • a parenteral agent examples include an injection, inhalation powders, an inhalation liquid, eye drops, a liquid agent, a lotion agent, a spray agent, nasal drops, a drip, an ointment, a suppository, and a patch.
  • the pharmaceutical composition of the present invention is administered by various administration methods, depending on usage.
  • Examples of the administration method include oral administration, intravenous administration, intraperitoneal administration, subcutaneous administration, topical administration, and intramuscular administration.
  • the pharmaceutical composition of the present invention is prepared using the protein of the present invention and at least one pharmaceutical product additive.
  • the pharmaceutical composition of the present invention can be prepared by a known pharmaceutical method, depending on its dosage form.
  • examples of such a pharmaceutical product additive include an excipient, a disintegrator, a binder, a lubricant, a diluent, a buffer, a tonicity agent, a preservative, a stabilizer, and a solubilizer.
  • the above-described pharmaceutical product additive also includes a physiological saline and water for injection.
  • the pharmaceutical composition of the present invention can be prepared by being mixed with, being diluted with, or being dissolved in the above-described pharmaceutical product additives.
  • the dose of the protein of the present invention is determined, as appropriate, depending on the age, sex, and body weight of a patient, the degree of disease, a dosage form, an administration route, etc.
  • the applied dose can be determined, for example, in the range of 0.1 ⁇ g/kg to 1000 mg/kg/day.
  • the daily dose in the case of oral administration is preferably in the range of 0.1 mg/kg to 10 mg/kg/day, depending on the dosage form.
  • Such a daily dose may be administered at once, or dividedly over two or three administrations.
  • the dose applied to an adult by parenteral administration can be determined in the range of 0.01 ⁇ g/kg to 1000 mg/kg/day.
  • the daily dose in the case of parenteral administration is in the range of preferably 0.1 ⁇ g/kg to 10 ⁇ g/kg/day, 1 ⁇ g/kg to 100 ⁇ g/kg/day, or 10 ⁇ g/kg to 1000 ⁇ g/kg/day, depending on the dosage form.
  • cDNA encoding the amino acid sequence shown in SEQ ID NO: 3 was incorporated into a mammalian expression plasmid vector to prepare a Protein 1 expression plasmid.
  • the Protein 1 expression plasmid was introduced into Chinese hamster ovary cells (CHO) to establish a Protein 1 stable expression cell line. Secretion of Protein 1 into a culture supernatant was confirmed by SDS-PAGE.
  • Protein 1 was dissolved in a coating buffer, and an aliquot of the obtained solution was added onto a microplate.
  • the microplate was left at 4° C. for 18 or more hours, and was then washed with a washing buffer (PBS-Tween 20). Thereafter, a blocking solution (Assay Diluent) (BD Biosciences) was added thereto.
  • the microplate was left at room temperature for 1 hour, and the blocking solution was then removed. The plate was washed with a washing buffer, and was then used in the measurement of binding inhibitory activity.
  • omalizumab anti-human IgE antibody
  • the IgE binding inhibitory activity of Protein 1 was measured by the following method.
  • a certain amount of human IgE (ANTIBODYSHOP) was mixed with Protein 1 or omalizumab (Novartis) having any given concentration. The obtained mixture was added to the plate prepared in the above (1), and it was then left at room temperature for approximately 2 hours. After the mixed solution had been discarded, the plate was washed with a washing buffer. A HRP-labeled anti-human IgE antibody (BD Biosciences) was added to the resulting plate, and it was then left at room temperature for approximately 1 hour. After the antibody solution had been discarded, the plate was washed with a washing buffer. A TMB (3,3′,5,5′-tetramethylbenzidine) solution was added to the plate.
  • the Protein 1 of the present invention inhibited the binding of IgE to FCER1A in a concentration-dependent manner.
  • a purification operation was carried out using AKTA Explorer 10 S (GE Healthcare). Protein 1 was allowed to be expressed by the same method as that described in Example 1, and then, a culture supernatant thereof was 2 times diluted with D-PBS( ⁇ ) (Dulbecco's Phosphate Buffered Saline). The diluted solution was loaded on HiTrap rProtein A FF (GE Healthcare, 17-5079-01). The above-described column was washed with D-PBS( ⁇ ), and was then eluted with a 100 mM glycine-HCI buffer (pH 2.2). A protein A adsorption fraction was fractionated at 1.0 mL/tube.
  • D-PBS( ⁇ ) Dulbecco's Phosphate Buffered Saline
  • HiTrap rProtein A FF GE Healthcare, 17-5079-01
  • the above-described column was washed with D-PBS( ⁇ ), and was then eluted with a 100 mM g
  • the peak fractions were mixed with one another to obtain a low pH-treated sample (pH 2.9).
  • the low pH-treated sample that had been preserved at 4° C. was used as an evaluation sample.
  • 0.25 mL of an aliquot was sampled from the evaluation sample at a predetermined timing (5 to 12 days after the preservation at 4° C.), and 0.05 mL of a 1 M Tris-HCI buffer (pH 9.0) was added to the aliquot to neutralize it, thereby obtaining a neutralized sample.
  • Fusion protein A described in Non Patent Literature 3 was used as a control. Fusion protein A was allowed to be expressed by the same method as that described in Example 1, and then, a neutralized sample was obtained by the same method as the aforementioned method. It is to be noted that the Fusion protein A used in the present test is a protein consisting of the amino acid sequence shown in the following SEQ ID NO: 4.
  • the amino acid sequence shown in SEQ ID NO: 4 comprises a linker fragment consisting of Asp at position 180 to Cys at position 188, and the number of Cys residues contained in this linker fragment is 2.
  • SEQ ID NO : 4 VPQKPKVSLNPPWNRIFKGENVTLTCNGNNFFEVSSTKWFHNGSLSEETN SSLNIVNAKFEDSGEYKCQHQQVNESEPVYLEVFSDWLLLQASAEVVMEG QPLFLRCHGWRNWDVYKVIYYKDGEALKYWYENHNISITNATVEDSGTYY CTGKVWQLDYESEPLNITVIKAPREKYWLDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLS
  • the content of aggregates was confirmed by performing gel filtration chromatography of using Superdex 200 10/300GL (GE Healthcare, 17-5175-01), employing AKTA Explorer 10 S (GE Healthcare), and also using D-PBS( ⁇ ) as a mobile phase.
  • the areas of a peak whose eluted position was a monomer and an aggregate peak eluted in a high molecular region were calculated, and a transition of a change in the content rate of aggregates (%) in Protein 1 and Fusion protein A, which was caused by a low pH treatment, was evaluated ( FIG. 2 ).
  • the increased level of the formation of aggregates associated with a low pH treatment time was significantly smaller than that in the case of Fusion protein A, and thus, the protein of the present invention exhibited high stability against exposure to low pH. Therefore, the protein of the present invention is excellent in terms of stability at low pH, and thus, the improvement of purification efficiency and productivity is expected in the production process.
  • a purification operation was carried out using AKTA Explorer 10 S (GE Healthcare). Protein 1 was allowed to be expressed by the same method as that described in Example 1, and a culture supernatant thereof was then loaded on HiTrap Mab Select SuRe (GE Healthcare, 17-0034-94). The above-described column was washed with D-PBS( ⁇ ) and a 100 mM citrate buffer (pH 4.0), and a protein A adsorbate was then eluted with a 100 mM glycine-HCI buffer (pH 3.3). A 1 M Tris-HCI buffer (pH 9.0) was added at a volume of 1/10 to the recovered fraction to neutralize it, so as to obtain a protein A-purified protein.
  • A-purified protein was adjusted to pH 4.0 by addition of 1 N HCI, and it was then loaded on a column filled with a mixed mode resin of hydrophobic interaction and cation exchange.
  • a non-adsorbed protein was washed with a 50 mM acetate buffer (pH 4.0), and 100% linear gradient elution was then carried out with a 50 mM Tris-Hcl buffer (pH 9.0).
  • a peak fraction was recovered to obtain a purified protein.
  • the obtained protein was subjected to gel filtration fractionation, using D-PBS( ⁇ ) as a mobile phase, and employing HiLoad 16/60 Superdex 200 prep grade (GE Healthcare, 17-1069-01).
  • a peak fraction corresponding to a monomer was recovered to obtain a gel filtration purified sample.
  • This gel filtration purified sample was adjusted again with D-PBS( ⁇ ), and it was then poured into a microtube, followed by incubation at 37° C., so as to obtain an evaluation sample. An aliquot was sampled from this evaluation sample at a predetermined timing (1 day to 7 days after the preservation at 37° C.), so as to obtain a heat treatment sample.
  • Fusion protein A described in Non Patent Literature 3 was used as a control. Fusion protein A was allowed to be expressed by the same method as that in Example 1, and then, a heat treatment sample was obtained by the same method as the aforementioned method. It is to be noted that the Fusion protein A used in the present test is a protein consisting of the same amino acid sequence as that of the protein used in Test Example 2.
  • the content of aggregates was confirmed by performing gel filtration chromatography of using Superdex 200 10/300GL (GE Healthcare, 17-5175-01), employing AKTA Explorer 10 S (GE Healthcare), and also using D-PBS( ⁇ ) as a mobile phase.
  • the areas of a peak whose eluted position was a monomer and an aggregate peak eluted in a high molecular region were calculated, and a transition of a change in the content rate of aggregates (%) in Protein 1 and Fusion protein A, which was caused by a heat treatment, was evaluated ( FIG. 3 ).
  • the increased level of the content of aggregates after preservation at 37° C. was smaller than that in the case of Fusion protein A, and thus, the protein of the present invention exhibited more stability against exposure at 37° C. Therefore, the protein of the present invention is excellent in terms of stability against heat, as well as stability at low pH, and thus, the improvement of purification efficiency and productivity is expected in the production process.
  • the protein of the present invention has excellent neutralizing activity against IgE, it can be used as a protein pharmaceutical product for preventing or treating various diseases mediated by the IgE.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Cell Biology (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Provided is an Fc fusion high affinity IgE receptor α-chain having excellent stability at low pH. An Fc fusion protein comprising:
    • (i) a high affinity IgE receptor α-chain; and
    • (ii) an Fc region of IgG1, wherein
    • a linker fragment region between the (i) and the (ii) is the amino acid sequence shown in SEQ ID NO: 2.

Description

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • This application is a Continuation of U.S. application Ser. No. 15/552,065, which is a National Stage application of PCT/JP2016/054854, filed Feb. 19, 2016, which claims priority from Japanese application nos. JP 2015-032231, filed Feb. 20, 2015, and JP 2015-252231, filed Dec. 24, 2015.
  • The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-WEB and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jul. 10, 2018, is named sequence.txt and is 10 KB.
    • TECHNICAL FIELD
  • The present invention relates to an Fc fusion high affinity IgE receptor α-chain that is useful as a pharmaceutical product.
  • More specifically, the present invention relates to an Fc fusion high affinity IgE receptor α-chain having excellent stability at low pH, and a medicinal use thereof.
    • BACKGROUND ART
  • Immunoglobulin E (IgE) is one of an immunoglobulin group, which plays a role in allergic reactions. The IgE, which is secreted from B cells or is expressed on the surface of the B cells, binds to a high affinity IgE receptor (FcεRI) found on the surface of mast cells, basophils, etc. When an antigenic protein binds to IgE on a mast cell surface receptor, the IgE becomes a form in which it crosslinks the antigen. Thereafter, chemical mediators such as histamine or serotonin, which are stored in intracellular granules, are released. Consequently, an inflammatory reaction is induced, and type I allergy symptoms such as telangiectasis or vascular hyperpermeability are provoked (Non Patent Literature 1).
  • Accordingly, since a compound or a protein, which inhibits the binding of IgE to FcεRI, inhibits the binding of the IgE to the FcεRI found on the surface of mast cells, basophils, etc., such a compound or a protein is expected as a therapeutic agent for type I allergic diseases such as bronchial asthma, allergic rhinitis, and allergic conjunctivitis (Non Patent Literature 2).
  • In recent years, not only a conventional pharmaceutical product comprising, as an active ingredient, a low-molecular-weight compound, but also a protein pharmaceutical product, which strongly binds to a specific receptor or the like in a living body and exhibits excellent therapeutic effects, has been developed. For example, Etanercept has been known as a therapeutic agent for rheumatoid arthritis. Such Etanercept is a completely humanized soluble TNFα/LTα receptor formulation, which has been focused because of the role of a soluble receptor of a tumor necrosis factor (TNF) to suppress the action of TNF in a living body, and has been then developed.
  • The protein pharmaceutical product can be expected to have high therapeutic effects. On the other hand, it may cause a problem specific to the protein pharmaceutical product in the production process thereof.
  • In general, when an antibody or an Fc fusion protein is produced in the form of a pharmaceutical product, a purification method of using protein A is applied. In this method, a buffer with a low pH value is used to elute a protein of interest that binds to the protein A. Moreover, in order to inactivate virus, the protein of interest is desirably treated at low pH for a certain period of time.
  • A protein, which is poor in stability at low pH, easily forms aggregates. If the ratio of aggregates is high, a reduction in purification efficiency or production amount occurs in the production of protein pharmaceutical products. In addition, an immune response is provoked by mixing aggregates into pharmaceutical products, and as a result, serious side effects, such as anaphylaxis, are likely to occur.
  • As such, the instability of a protein of interest at low pH may be problematic in the production of protein pharmaceutical products.
  • A polypeptide (immunoadheson) comprising an immunoglobulin and an extracellular domain is disclosed in Patent Literature 1. Patent Literature 1 discloses a high affinity IgE receptor as an example of such immunoadheson. However, this publication does not specifically describe a fused protein of a high affinity IgE receptor and an immunoglobulin.
  • A fused protein (hereinafter referred to as “Fusion protein A”) of a high affinity IgE receptor α-chain (FcεRI α-chain; hereinafter referred to as “FCER1A”) and immunoglobulin G1 (IgG1) is disclosed in Non Patent Literature 3. However, the Fusion protein A disclosed in the aforementioned publication is largely different from the protein of the invention of the present application, in terms of the mode of binding FCER1A to IgG1 (Fc). That is to say, the protein of the invention of the present application has a characteristic amino acid sequence in a linker fragment region between the FcεRI and the IgG1.
  • A fused protein (NPB301) formed by linking a water-soluble fragment of the high affinity IgE receptor (FcεRI) to a human Fc region via a peptide linker is disclosed in Patent Literature 2. However, the protein of the invention of the present application does not comprise the peptide linker disclosed in Patent Literature 2. Moreover, Patent Literature 2 neither discloses nor suggests the characteristic amino acid sequence of the linker fragment region of the present invention.
  • A fused protein of FCER1A and immunoglobulin G2 (IgG2) is disclosed in Patent Literature 3. However, this fused protein comprising IgG2 is different from the protein of the invention of the present application, in terms of the amino acid sequences of a linker fragment region and an Fc region.
  • A fused protein of non-human primate FCER1A and IgG1 is disclosed in Patent Literature 4. Moreover, a fused protein of FCER1A and IgG1 is disclosed in Patent Literatures 5 to 7. However, these publications neither disclose nor suggest the characteristic amino acid sequence of the linker fragment region of the present invention.
  • The aforementioned Non Patent Literature 3 and Patent Literatures 1 to 7 neither disclose nor suggest the protein of the present invention.
    • CITATION LIST Patent Literature
    • Patent Literature 1: U.S. Pat. No. 5,565,335
    • Patent Literature 2: International Publication WO 2012/169735
    • Patent Literature 3: Chinese Patent Application Laid-Open No. 101633698
    • Patent Literature 4: International Publication WO 2008/028068
    • Patent Literature 5: International Publication WO 2011/056606
    • Patent Literature 6: International Publication WO 2008/099178
    • Patent Literature 7: International Publication WO 2008/099188
    Non Patent Literature
    • Non Patent Literature 1: Chisei Ra, “Allergy no Bunshi Saibo Kiko (Molecular and Cellular Mechanisms of Allergy),” BIO INDUSTRY, 2008, Vol. 25, No. 9, PP. 23-39
    • Non Patent Literature 2: Chisei Ra et al., “International Immunology,” 1993, Vol. 5, No. 1, PP. 47-54
    • Non Patent Literature 3: M. Haak-Frendscho et al., “Journal of Immunology,” 1993, Vol. 151, No. 1, PP. 351-358
    SUMMARY OF INVENTION Technical Problem
  • It is an object of the present invention to provide an Fc fusion high affinity IgE receptor α-chain having excellent stability at low pH.
    • Solution to Problem
  • The present inventors have conducted intensive studies in order to obtain an Fc fusion high affinity IgE receptor α-chain having high stability against low pH or heat. As a result, the inventors have found that an Fc fusion high affinity IgE receptor α-chain having high stability can be obtained by using a linker fragment comprising three Cys residues, in a fused protein comprising a high affinity IgE receptor α-chain and the Fc region of IgG1, thereby completing the present invention. Specifically, the present invention is as follows.
  • The present invention relates to the following [1] to [5], etc.
    • [1] An Fc fusion protein comprising:
  • (i) a high affinity IgE receptor α-chain; and
  • (ii) an Fc region of IgG1, wherein
  • a linker fragment region between the (i) and the (ii) is the amino acid sequence shown in SEQ ID NO: 2.
    • [2] The Fc fusion protein according to the above [1], comprising the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence comprising a deletion of lysine (K) at the C-terminus of the amino acid sequence shown in SEQ ID NO: 3.
    • [3] The Fc fusion protein according to the above [1] or [2], which is a dimer.
    • [4] The Fc fusion protein according to the above [3], wherein cysteine residues in the linker fragment region form three disulfide bonds.
    • [5] A pharmaceutical composition comprising, as an active ingredient, the Fc fusion protein according to any one of the above [1] to [4].
  • The present description includes the contents as disclosed in Japanese Patent Application No. 2015-032231 and 2015-252231, which are priority documents of the present application.
    • Advantageous Effects of Invention
  • The protein of the present invention has excellent stability at low pH. In addition, the protein of the present invention has excellent neutralizing activity against IgE. Accordingly, the protein of the present invention is useful as a protein pharmaceutical product for preventing or treating type I allergic diseases mediated by IgE.
    • BRIEF DESCRIPTION OF DRAWINGS
  • FIG. 1 shows the activity of inhibiting the binding of human IgE. In the figure, the horizontal axis indicates the concentration of each drug (mol/L); and the longitudinal axis indicates the value of the amount of IgE bound to Protein 1 immobilized on a plate, which is shown at a percentage (free IgE (percent with respect to a control)), using, as a reference, the binding amount obtained when a predetermined amount of IgE is added. In the figure, the circle indicates the value of Protein 1, and the square indicates the value of Omalizumab, respectively.
  • FIG. 2 shows a transition of a change in the content rate (%) of aggregates at low pH. In the figure, the horizontal axis indicates the number of days (Day), and the longitudinal axis indicates a change in the content rate (%) of aggregates. In the figure, the circle indicates the value of Protein 1, and the square indicates the value of Fusion protein A, respectively.
  • FIG. 3 shows a transition of a change in the content rate (%) of aggregates by a heat treatment. In the figure, the horizontal axis indicates the number of days (Day), and the longitudinal axis indicates a change in the content rate (%) of aggregates. In the figure, the circle indicates the value of Protein 1, and the square indicates the value of Fusion protein A, respectively.
    •  DESCRIPTION OF EMBODIMENTS
  • The embodiments of the present invention will be described in detail below.
  • In the present invention, individual terms have the following meanings, unless otherwise specified.
  • In the present invention, the “high affinity IgE receptor α-chain (FCER1A)” means a protein comprising an α-chain portion that is an extracellular domain of a high affinity IgE receptor. The high affinity IgE receptor α-chain is, for example, a protein shown in the following SEQ ID NO: 1.
  • SEQ ID NO: 1:
    VPQKPKVSLNPPWNRIFKGENVTLTCNGNNFFEVSSTKWFHNGSLSEETN
    SSLNIVNAKFEDSGEYKCQHQQVNESEPVYLEVFSDWLLLQASAEVVMEG
    QPLFLRCHGWRNWDVYKVIYYKDGEALKYWYENHNISITNATVEDSGTYY
    CTGKVWQLDYESEPLNITVIKAPREKYWL
  • The above-described high affinity IgE receptor α-chain includes, for example, a protein having identity of 90% or more, 95% or more, 97% or more, or 99% or more to the amino acid sequence shown in SEQ ID NO: 1, when the identity is calculated using BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) or the like (e.g., using default parameters), and having binding ability to IgE. Moreover, the high affinity IgE receptor α-chain also includes a protein comprising an amino acid sequence having a substitution, deletion and/or addition of one or more, or several amino acids (1 to 10, preferably 1 to 5, and more preferably 1 or 2 amino acids) with respect to the amino acid sequence shown in SEQ ID NO: 1, and having binding ability to IgE.
  • In the present invention, the “Fc region of IgG1” means the Fc fragment of immunoglobulin G1, namely, the CH2 and CH3 constant domains of native immunoglobulin G1. The Fc region of IgG1 includes all of a natural mutant, an artificial mutant, and a truncated form.
  • In the present invention, the “linker fragment region between the high affinity IgE receptor α-chain and the Fc region of IgG1” means a region consisting of 14 amino acid residues ranging from the junction point between the above-described high affinity IgE receptor α-chain and the above described Fc region of IgG1 towards the direction of the Fc region.
  • In the present invention, the “Fc fusion protein” means a recombinant protein comprising the high affinity IgE receptor α-chain and the Fc fragment of an immunoglobulin.
  • The protein of the present invention is characterized in that the linker fragment region between the high affinity IgE receptor α-chain and the Fc region of IgG1 is the amino acid sequence shown in the following SEQ ID NO: 2.
  • SEQ ID NO: 2:
    EPKSCDKTHTCPPC
  • The protein of the present invention is preferably an Fc fusion protein comprising the amino acid sequence shown in the following SEQ ID NO: 3 (hereinafter referred to as “Protein 1”).
  • SEQ ID NO: 3:
    VPQKPKVSLNPPWNRIFKGENVTLTCNGNNFFEVSSTKWFHNGSLSEETN
    SSLNIVNAKFEDSGEYKCQHQQVNESEPVYLEVFSDWLLLQASAEVVMEG
    QPLFLRCHGWRNWDVYKVIYYKDGEALKYWYENHNISITNATVEDSGTYY
    CTGKVWQLDYESEPLNITVIKAPREKYWLEPKSCDKTHTCPPCPAPELLG
    GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN
    AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI
    SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ
    PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFScSVMHEALHNHY
    TQKSLSLSPGK
  • The amino acid sequence shown in SEQ ID NO: 3 has a sequence formed by fusing the amino acid sequence shown in SEQ ID NO: 1 (from Val at position 1 to Leu at position 179 of the amino acid sequence shown in SEQ ID NO: 3), the amino acid sequence shown in SEQ ID NO: 2 (Glu at position 180 to Cys at position 193 of the amino acid sequence shown in SEQ ID NO: 3), and the amino acid sequence of the Fc fragment of an immunoglobulin (Pro at position 194 to Lys 411 of the amino acid sequence shown in SEQ ID NO: 3) in this order. This Fc fusion protein includes a protein comprising an amino acid sequence having identity of 90% or more, 95% or more, 97% or more, or 99% or more to the amino acid sequence shown in SEQ ID NO: 3, which is other than the amino acid sequence portion from Glu at position 180 to Cys at position 193 that corresponds to the amino acid sequence shown in SEQ ID NO: 2, when the identity is calculated using, for example, BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) or the like (e.g., using default parameters), and having binding ability to IgE. Moreover, this Fc fusion protein includes a protein comprising an amino acid sequence having a substitution, deletion and/or addition of one or more, or several amino acids (1 to 10, preferably 1 to 5, and more preferably 1 or 2 amino acids) with respect to the amino acid sequence shown in SEQ ID NO: 3, which is other than the amino acid sequence portion from Glu at position 180 to Cys at position 193 that corresponds to the amino acid sequence shown in SEQ ID NO: 2, and having binding ability to IgE.
  • Upon the production of a recombinant antibody, there is a case where the lysine at the C-terminus is deleted by post-translational modification. Accordingly, the protein of the present invention may be an Fc fusion protein comprising an amino acid sequence having a deletion of the lysine (K) at the C-terminus of the above-described Protein 1. For example, such an Fc fusion protein comprising an amino acid sequence having a deletion of the lysine (K) at the C-terminus of Protein 1 shown in SEQ ID NO: 3 consists of an amino acid sequence portion from the position 1 to the position 410 of the amino acid sequence shown in SEQ ID NO: 3.
  • The protein of the present invention includes both a monomer and a dimer of an Fc fusion protein formed by linking a high affinity IgE receptor α-chain to the Fc region of IgG1 via a linker fragment consisting of the amino acid sequence shown in SEQ ID NO: 2. Three Cys residues are present in the above-described linker fragment region (the Cys residues at positions 184, 190, and 193 of the amino acid sequence shown in SEQ ID NO: 3), and a dimer can be formed by disulfide bonds. In general, two Fc fusion protein monomers form a dimer as a result of the formation of three disulfide bonds between Cys residues at the same positions as those of the above-described three Cys residues, and the above-described three Cys residues. Because of the above-described three disulfide bonds, a dimer is stabilized, and it has high stability against low pH and heat. The phrase “having high stability against low pH and heat” means, for example, that only a few aggregates are formed under conditions of low pH and under heating. Such high stability against low pH or heat can be confirmed, for example, by performing a low pH treatment or a heat treatment on a protein, and then measuring the content of aggregates in the protein by gel filtration chromatography. For example, even if the Fc fusion protein of the present invention is preserved at a low temperature of 2° C. to 8° C., and preferably 4° C., at pH 1 to 5, and preferably at pH 2 to 4, for 1 day to 1 month, preferably for 1 day to 14 days, and more preferably for 5 to 12 days, or even if the present Fc fusion protein is preserved at a temperature of 25° C. to 45° C., and preferably 30° C. to 40° C., for 1 day to 1 month, preferably 1 day to 14 days, and more preferably 1 day to 7 days, a change in the content rate of aggregates is small. For example, when a change in the content rate of aggregates in the protein of the present invention is calculated based on the peak area of gel filtration chromatography, it is 10% or less, and preferably 8% or less. Further, when compared with an Fc fusion protein having two or less Cys residues as a linker fragment, a change in the content rate of aggregates in the protein of the present invention is small.
  • The protein of the present invention can be produced, for example, by the following method or a method equivalent thereto, or by the methods described in publications or methods equivalent thereto.
  • The protein of the present invention can be produced using a genetic recombination technique that is well known to a person skilled in the present technical field. For example, DNA encoding the protein of the present invention is prepared, and an expression vector comprising this DNA is then constructed. Subsequently, prokaryotic or eukaryotic cells are transformed or transfected with the above-described vector, and a protein of interest can be isolated and purified from a culture supernatant of the obtained cells.
  • The protein of the present invention can also be produced using protein-expressing cells that are well known to a person skilled in the present technical field. For example, cDNA encoding the amino acid sequence shown in SEQ ID NO: 3 is incorporated into a mammalian expression plasmid vector to prepare a protein expression plasmid, and the prepared plasmid is then introduced into animal cells such as Chinese hamster ovary cells (CHO), so as to establish a stable expression cell line. The obtained cells are cultured, and then, the protein of the present invention can be obtained from a culture supernatant.
  • The protein of the present invention can be isolated and purified, as necessary, by isolation and purification means that are well known to a person skilled in the present technical field. Examples of the isolation and purification method include affinity chromatography, ion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, mixed mode chromatography, dialysis, a fractional precipitation method, and electrophoresis. The protein of the present invention can also be isolated and purified by combining these methods with one another, as appropriate.
  • The protein of the present invention may also undergo chemical modification that is well known to a person skilled in the present technical field. Examples of the chemical modification include glycosylation, polyethylene glycolation (PEG), acetylation, and amidation.
  • Since the protein of the present invention has neutralizing activity against IgE, it can be used as a preventive or therapeutic agent for various diseases mediated by IgE. For instance, the protein of the invention of the present application is useful as a preventive or therapeutic agent for diseases associated with type I allergy, and the like, such as bronchial asthma, eosinophilic otitis media, eosinophilic sinusitis, allergic conjunctivitis, allergic rhinitis, pollinosis, food allergy, mite allergic disease, hives, and anaphylactic shock.
  • The protein of the present invention has excellent affinity for IgE. Hence, the protein of the present invention can also be used as a “protein-drug conjugate” that utilizes such affinity, such as an antibody-drug conjugate (ADC). Examples of such a “protein-drug conjugate” include the modes of use, such as “Protein 1-drug” and “Protein 1-linker-drug.” As a drug, an anti-allergic agent or the like can be used. Such a conjugate can be produced by a method well known to a person skilled in the present technical field.
  • With regard to the pharmaceutical composition of the present invention, various dosage forms are used depending on usage. Examples of an oral agent include a tablet, a powder agent, a granule, a fine granule, and a capsule. Examples of a parenteral agent include an injection, inhalation powders, an inhalation liquid, eye drops, a liquid agent, a lotion agent, a spray agent, nasal drops, a drip, an ointment, a suppository, and a patch.
  • The pharmaceutical composition of the present invention is administered by various administration methods, depending on usage. Examples of the administration method include oral administration, intravenous administration, intraperitoneal administration, subcutaneous administration, topical administration, and intramuscular administration.
  • The pharmaceutical composition of the present invention is prepared using the protein of the present invention and at least one pharmaceutical product additive. The pharmaceutical composition of the present invention can be prepared by a known pharmaceutical method, depending on its dosage form. Examples of such a pharmaceutical product additive include an excipient, a disintegrator, a binder, a lubricant, a diluent, a buffer, a tonicity agent, a preservative, a stabilizer, and a solubilizer. The above-described pharmaceutical product additive also includes a physiological saline and water for injection. The pharmaceutical composition of the present invention can be prepared by being mixed with, being diluted with, or being dissolved in the above-described pharmaceutical product additives.
  • When the pharmaceutical composition of the present invention is used for prevention or treatment, the dose of the protein of the present invention, which is comprised therein as an active ingredient, is determined, as appropriate, depending on the age, sex, and body weight of a patient, the degree of disease, a dosage form, an administration route, etc. With regard to the dose applied to an adult by oral administration, the applied dose can be determined, for example, in the range of 0.1 μg/kg to 1000 mg/kg/day. The daily dose in the case of oral administration is preferably in the range of 0.1 mg/kg to 10 mg/kg/day, depending on the dosage form. Such a daily dose may be administered at once, or dividedly over two or three administrations. Moreover, the dose applied to an adult by parenteral administration can be determined in the range of 0.01 μg/kg to 1000 mg/kg/day. The daily dose in the case of parenteral administration is in the range of preferably 0.1 μg/kg to 10 μg/kg/day, 1 μg/kg to 100 μg/kg/day, or 10 μg/kg to 1000 μg/kg/day, depending on the dosage form.
    • EXAMPLES
  • The content of the present invention will be described in more detail in the following examples and test examples. However, these examples are not intended to limit the scope of the present invention.
    • Example 1 Expression and Preparation of Protein 1 (1) Preparation of Protein 1 Expression Vector
  • cDNA encoding the amino acid sequence shown in SEQ ID NO: 3 was incorporated into a mammalian expression plasmid vector to prepare a Protein 1 expression plasmid.
    • (2) Preparation of Protein 1-Expressing Cells
  • The Protein 1 expression plasmid was introduced into Chinese hamster ovary cells (CHO) to establish a Protein 1 stable expression cell line. Secretion of Protein 1 into a culture supernatant was confirmed by SDS-PAGE.
    • Test Example 1 IgE Binding Inhibitory Activity (IgE Neutralizing Activity) (1) Preparation of Assay Plate
  • Protein 1 was dissolved in a coating buffer, and an aliquot of the obtained solution was added onto a microplate. The microplate was left at 4° C. for 18 or more hours, and was then washed with a washing buffer (PBS-Tween 20). Thereafter, a blocking solution (Assay Diluent) (BD Biosciences) was added thereto. The microplate was left at room temperature for 1 hour, and the blocking solution was then removed. The plate was washed with a washing buffer, and was then used in the measurement of binding inhibitory activity.
    • (2) Method of Measuring Binding Inhibitory Activity
  • Using omalizumab (anti-human IgE antibody) as a positive control, the IgE binding inhibitory activity of Protein 1 was measured by the following method.
  • A certain amount of human IgE (ANTIBODYSHOP) was mixed with Protein 1 or omalizumab (Novartis) having any given concentration. The obtained mixture was added to the plate prepared in the above (1), and it was then left at room temperature for approximately 2 hours. After the mixed solution had been discarded, the plate was washed with a washing buffer. A HRP-labeled anti-human IgE antibody (BD Biosciences) was added to the resulting plate, and it was then left at room temperature for approximately 1 hour. After the antibody solution had been discarded, the plate was washed with a washing buffer. A TMB (3,3′,5,5′-tetramethylbenzidine) solution was added to the plate. After a certain period of time had passed, phosphoric acid was added to the plate to terminate a coloring reaction. Thereafter, using a plate reader, absorbance (OD450) was measured. Based on the amount of IgE bound to Protein 1 immobilized on the plate, Protein 1 and omalizumab were evaluated in terms of IgE binding inhibitory activity (IgE neutralizing activity) (FIG. 1).
    • (3) Results
  • The Protein 1 of the present invention inhibited the binding of IgE to FCER1A in a concentration-dependent manner.
    • Test Example 2 Test Regarding Stability at Low pH (1) Preparation of Sample
  • A purification operation was carried out using AKTA Explorer 10 S (GE Healthcare). Protein 1 was allowed to be expressed by the same method as that described in Example 1, and then, a culture supernatant thereof was 2 times diluted with D-PBS(−) (Dulbecco's Phosphate Buffered Saline). The diluted solution was loaded on HiTrap rProtein A FF (GE Healthcare, 17-5079-01). The above-described column was washed with D-PBS(−), and was then eluted with a 100 mM glycine-HCI buffer (pH 2.2). A protein A adsorption fraction was fractionated at 1.0 mL/tube. The peak fractions were mixed with one another to obtain a low pH-treated sample (pH 2.9). The low pH-treated sample that had been preserved at 4° C. was used as an evaluation sample. 0.25 mL of an aliquot was sampled from the evaluation sample at a predetermined timing (5 to 12 days after the preservation at 4° C.), and 0.05 mL of a 1 M Tris-HCI buffer (pH 9.0) was added to the aliquot to neutralize it, thereby obtaining a neutralized sample.
  • As a control, Fusion protein A described in Non Patent Literature 3 was used. Fusion protein A was allowed to be expressed by the same method as that described in Example 1, and then, a neutralized sample was obtained by the same method as the aforementioned method. It is to be noted that the Fusion protein A used in the present test is a protein consisting of the amino acid sequence shown in the following SEQ ID NO: 4. The amino acid sequence shown in SEQ ID NO: 4 comprises a linker fragment consisting of Asp at position 180 to Cys at position 188, and the number of Cys residues contained in this linker fragment is 2.
  • SEQ ID NO : 4:
    VPQKPKVSLNPPWNRIFKGENVTLTCNGNNFFEVSSTKWFHNGSLSEETN
    SSLNIVNAKFEDSGEYKCQHQQVNESEPVYLEVFSDWLLLQASAEVVMEG
    QPLFLRCHGWRNWDVYKVIYYKDGEALKYWYENHNISITNATVEDSGTYY
    CTGKVWQLDYESEPLNITVIKAPREKYWLDKTHTCPPCPAPELLGGPSVF
    LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
    REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
    QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
    KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL
    SLSPGK
    • (2) Method of Analyzing Content of Aggregates
  • With regard to the operation of analyzing aggregates, the content of aggregates was confirmed by performing gel filtration chromatography of using Superdex 200 10/300GL (GE Healthcare, 17-5175-01), employing AKTA Explorer 10 S (GE Healthcare), and also using D-PBS(−) as a mobile phase. The areas of a peak whose eluted position was a monomer and an aggregate peak eluted in a high molecular region were calculated, and a transition of a change in the content rate of aggregates (%) in Protein 1 and Fusion protein A, which was caused by a low pH treatment, was evaluated (FIG. 2).
    • (3) Results
  • In the protein of the present invention, the increased level of the formation of aggregates associated with a low pH treatment time was significantly smaller than that in the case of Fusion protein A, and thus, the protein of the present invention exhibited high stability against exposure to low pH. Therefore, the protein of the present invention is excellent in terms of stability at low pH, and thus, the improvement of purification efficiency and productivity is expected in the production process.
    • Test Example 3 Test Regarding Stability Against Heat (1) Preparation of Sample
  • A purification operation was carried out using AKTA Explorer 10 S (GE Healthcare). Protein 1 was allowed to be expressed by the same method as that described in Example 1, and a culture supernatant thereof was then loaded on HiTrap Mab Select SuRe (GE Healthcare, 17-0034-94). The above-described column was washed with D-PBS(−) and a 100 mM citrate buffer (pH 4.0), and a protein A adsorbate was then eluted with a 100 mM glycine-HCI buffer (pH 3.3). A 1 M Tris-HCI buffer (pH 9.0) was added at a volume of 1/10 to the recovered fraction to neutralize it, so as to obtain a protein A-purified protein. The pH of this protein A-purified protein was adjusted to pH 4.0 by addition of 1 N HCI, and it was then loaded on a column filled with a mixed mode resin of hydrophobic interaction and cation exchange. A non-adsorbed protein was washed with a 50 mM acetate buffer (pH 4.0), and 100% linear gradient elution was then carried out with a 50 mM Tris-Hcl buffer (pH 9.0). A peak fraction was recovered to obtain a purified protein. The obtained protein was subjected to gel filtration fractionation, using D-PBS(−) as a mobile phase, and employing HiLoad 16/60 Superdex 200 prep grade (GE Healthcare, 17-1069-01). A peak fraction corresponding to a monomer was recovered to obtain a gel filtration purified sample. This gel filtration purified sample was adjusted again with D-PBS(−), and it was then poured into a microtube, followed by incubation at 37° C., so as to obtain an evaluation sample. An aliquot was sampled from this evaluation sample at a predetermined timing (1 day to 7 days after the preservation at 37° C.), so as to obtain a heat treatment sample.
  • As a control, Fusion protein A described in Non Patent Literature 3 was used. Fusion protein A was allowed to be expressed by the same method as that in Example 1, and then, a heat treatment sample was obtained by the same method as the aforementioned method. It is to be noted that the Fusion protein A used in the present test is a protein consisting of the same amino acid sequence as that of the protein used in Test Example 2.
    • (2) Method of Analyzing Content of Aggregates
  • With regard to the operation of analyzing aggregates, the content of aggregates was confirmed by performing gel filtration chromatography of using Superdex 200 10/300GL (GE Healthcare, 17-5175-01), employing AKTA Explorer 10 S (GE Healthcare), and also using D-PBS(−) as a mobile phase. The areas of a peak whose eluted position was a monomer and an aggregate peak eluted in a high molecular region were calculated, and a transition of a change in the content rate of aggregates (%) in Protein 1 and Fusion protein A, which was caused by a heat treatment, was evaluated (FIG. 3).
    • (3) Results
  • In the protein of the present invention, the increased level of the content of aggregates after preservation at 37° C. was smaller than that in the case of Fusion protein A, and thus, the protein of the present invention exhibited more stability against exposure at 37° C. Therefore, the protein of the present invention is excellent in terms of stability against heat, as well as stability at low pH, and thus, the improvement of purification efficiency and productivity is expected in the production process.
    • INDUSTRIAL APPLICABILITY
  • Since the protein of the present invention has excellent neutralizing activity against IgE, it can be used as a protein pharmaceutical product for preventing or treating various diseases mediated by the IgE.
    • SEQUENCING FREE TEXT
    • SEQ ID NO: 2 Synthesized
  • All publications, patents and patent applications cited in the present description are incorporated herein by reference in their entirety.

Claims (4)

1. An Fc fusion protein comprising:
(i) a high affinity IgE receptor α-chain; and
(ii) an Fc region of IgG1, wherein
a linker fragment region between the (i) and the (ii) is the amino acid sequence shown in SEQ ID NO: 2.
2. The Fc fusion protein according to claim 1, which is a dimer.
3. The Fc fusion protein according to claim 2, wherein cysteine residues in the linker fragment region form three disulfide bonds.
4. A pharmaceutical composition comprising, as an active ingredient, the Fc fusion protein according to claims 1.
US16/034,648 2015-02-20 2018-07-13 Fc FUSION HIGH AFFINITY IgE RECEPTOR ALPHA-CHAIN Abandoned US20180312566A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US16/034,648 US20180312566A1 (en) 2015-02-20 2018-07-13 Fc FUSION HIGH AFFINITY IgE RECEPTOR ALPHA-CHAIN

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
JP2015032231 2015-02-20
JP2015-032231 2015-02-20
JP2015252231 2015-12-24
JP2015-252231 2015-12-24
PCT/JP2016/054854 WO2016133197A1 (en) 2015-02-20 2016-02-19 Fc-FUSED HIGH AFFINITY IgE RECEPTOR α-CHAIN
US201715552065A 2017-08-18 2017-08-18
US16/034,648 US20180312566A1 (en) 2015-02-20 2018-07-13 Fc FUSION HIGH AFFINITY IgE RECEPTOR ALPHA-CHAIN

Related Parent Applications (2)

Application Number Title Priority Date Filing Date
PCT/JP2016/054854 Continuation WO2016133197A1 (en) 2015-02-20 2016-02-19 Fc-FUSED HIGH AFFINITY IgE RECEPTOR α-CHAIN
US15/552,065 Continuation US10077297B2 (en) 2015-02-20 2016-02-19 Fc fusion high affinity IgE receptor α-chain

Publications (1)

Publication Number Publication Date
US20180312566A1 true US20180312566A1 (en) 2018-11-01

Family

ID=56692248

Family Applications (2)

Application Number Title Priority Date Filing Date
US15/552,065 Active US10077297B2 (en) 2015-02-20 2016-02-19 Fc fusion high affinity IgE receptor α-chain
US16/034,648 Abandoned US20180312566A1 (en) 2015-02-20 2018-07-13 Fc FUSION HIGH AFFINITY IgE RECEPTOR ALPHA-CHAIN

Family Applications Before (1)

Application Number Title Priority Date Filing Date
US15/552,065 Active US10077297B2 (en) 2015-02-20 2016-02-19 Fc fusion high affinity IgE receptor α-chain

Country Status (19)

Country Link
US (2) US10077297B2 (en)
EP (1) EP3260469B1 (en)
JP (1) JP6192862B2 (en)
KR (2) KR102602539B1 (en)
CN (1) CN107207623B (en)
AU (1) AU2016220766B2 (en)
BR (1) BR112017017609A2 (en)
CA (1) CA2976623C (en)
ES (1) ES2806205T3 (en)
HK (1) HK1244016A1 (en)
IL (1) IL253832B (en)
MX (1) MX2017010359A (en)
PL (1) PL3260469T3 (en)
PT (1) PT3260469T (en)
RU (1) RU2715606C2 (en)
SG (1) SG11201706543UA (en)
TW (1) TWI691512B (en)
WO (1) WO2016133197A1 (en)
ZA (2) ZA201706281B (en)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3538558A4 (en) * 2016-11-09 2020-07-15 North Carolina State University Treatment of allergic diseases with chimeric protein
KR102038672B1 (en) * 2018-01-08 2019-10-30 (주)지아이이노베이션 PHARMACEUTICAL COMPOSITION COMPRISING RECOMBINANT EXTRACELLULAR DOMAIN OF IgE Fc RECEPTOR SUBUNIT ALPHA
CN118638245A (en) * 2018-01-08 2024-09-13 Gi 医诺微新 Extracellular domain of alpha subunit of IgE Fc receptor, pharmaceutical composition comprising same and preparation method thereof
BR112020013818A2 (en) * 2018-01-12 2020-12-01 Gi Innovation, Inc. composition comprising probiotics and polypeptide with binding affinity for ige and use thereof
WO2019183437A1 (en) 2018-03-23 2019-09-26 North Carolina State University Methods and compositions for antibody to high affinity receptor for ige
CN114080398B (en) * 2019-07-08 2024-05-31 Gi医诺微新 Polypeptide dimer comprising extracellular domain of alpha subunit of IgE Fc receptor and having high sialic acid content, and pharmaceutical composition comprising same
KR20210006300A (en) * 2019-07-08 2021-01-18 주식회사 프로젠 A novel pharmaceutical composition for treating allergy
WO2021006375A1 (en) * 2019-07-08 2021-01-14 (주)지아이이노베이션 Polypeptide dimer containing high amount of sialic acid and including extracellular domain of alpha subunit of ige fc receptor, and pharmaceutical composition containing same

Family Cites Families (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5336603A (en) 1987-10-02 1994-08-09 Genentech, Inc. CD4 adheson variants
US5641870A (en) * 1995-04-20 1997-06-24 Genentech, Inc. Low pH hydrophobic interaction chromatography for antibody purification
WO2003048334A2 (en) * 2001-12-04 2003-06-12 Merck Patent Gmbh Immunocytokines with modulated selectivity
WO2008028068A2 (en) * 2006-08-30 2008-03-06 Genentech, Inc. NON-HUMAN PRIMATE FCεR1α POLYPEPTIDES
JP2010519194A (en) 2007-02-15 2010-06-03 アストラゼネカ・アクチエボラーグ Binding elements for IgE molecules
AR065368A1 (en) 2007-02-15 2009-06-03 Astrazeneca Ab ANTIBODIES FOR IGE MOLECULES
ES2478242T3 (en) * 2007-03-22 2014-07-21 Genentech, Inc. Apoptotic anti-IgE antibodies that bind with membrane bound IgE
KR101631454B1 (en) 2008-10-31 2016-06-17 가부시키가이샤 한도오따이 에네루기 켄큐쇼 Logic circuit
CN101633698B (en) 2009-08-26 2011-12-21 北京精益泰翔技术发展有限公司 Immune fusion protein and gene encoding same and application thereof
JP6139885B2 (en) 2009-10-26 2017-05-31 ジェネンテック， インコーポレイテッド Assays for detecting antibodies specific for therapeutic anti-IgE antibodies and their use in anaphylaxis
ES2826894T3 (en) * 2010-02-19 2021-05-19 Xencor Inc New immunoadhesins CTLA4-IG
GB201014033D0 (en) * 2010-08-20 2010-10-06 Ucb Pharma Sa Biological products
WO2012169735A2 (en) 2011-06-07 2012-12-13 (주)네오팜 Complex comprising water-soluble fragments of fcεri and composition comprising same for treating allergic diseases mediated by ige
GB201203051D0 (en) * 2012-02-22 2012-04-04 Ucb Pharma Sa Biological products
GB201203071D0 (en) 2012-02-22 2012-04-04 Ucb Pharma Sa Biological products
WO2014087010A1 (en) 2012-12-07 2014-06-12 Ablynx N.V. IMPROVED POLYPEPTIDES DIRECTED AGAINST IgE

Also Published As

Publication number Publication date
RU2017132689A (en) 2019-03-20
ES2806205T3 (en) 2021-02-16
EP3260469B1 (en) 2020-05-13
JP6192862B2 (en) 2017-09-06
ZA201804191B (en) 2019-09-25
HK1244016A1 (en) 2018-07-27
US20180044402A1 (en) 2018-02-15
KR102602539B1 (en) 2023-11-15
EP3260469A1 (en) 2017-12-27
CN107207623A (en) 2017-09-26
TWI691512B (en) 2020-04-21
ZA201706281B (en) 2019-04-24
SG11201706543UA (en) 2017-09-28
WO2016133197A1 (en) 2016-08-25
IL253832A0 (en) 2017-09-28
KR20230132610A (en) 2023-09-15
RU2017132689A3 (en) 2019-08-15
KR20170120579A (en) 2017-10-31
CA2976623A1 (en) 2016-08-25
US10077297B2 (en) 2018-09-18
TW201636373A (en) 2016-10-16
PT3260469T (en) 2020-07-29
AU2016220766A1 (en) 2017-08-31
AU2016220766B2 (en) 2021-03-04
BR112017017609A2 (en) 2018-05-08
MX2017010359A (en) 2018-01-23
CA2976623C (en) 2023-03-21
RU2715606C2 (en) 2020-03-02
JPWO2016133197A1 (en) 2017-06-15
NZ734482A (en) 2022-03-25
IL253832B (en) 2021-05-31
EP3260469A4 (en) 2018-08-22
PL3260469T3 (en) 2020-11-02
CN107207623B (en) 2021-01-29

Similar Documents

Publication Publication Date Title
US10077297B2 (en) Fc fusion high affinity IgE receptor α-chain
JP6329585B2 (en) Monomeric antibody Fc
KR101637502B1 (en) Therapeutic canine immunoglobulins and methods of using the same
EP2964674B1 (en) Anti-tnf-anti-il-17 bispecific antibodies
CN110234355B (en) Monomeric human IgG1Fc and bispecific antibodies
US9708402B2 (en) Anti-BAFF-anti-IL-17 bispecific antibodies
JP2004536605A (en) Interleukin 1β antibody
EP3101035B1 (en) Bifunctional fusion protein, preparation method therefor, and use thereof
WO2023061390A1 (en) Treatment of ige-mediated diseases
NZ734482B2 (en) Fc FUSION HIGH AFFINITY IgE RECEPTOR α-CHAIN
CN112930358A (en) anti-TNF alpha/anti-IL-17A natural antibody structure-like heterodimer form bispecific antibody and preparation thereof
CN117279942A (en) Compounds and methods for treating pain

Legal Events

Date Code Title Description
STPP Information on status: patent application and granting procedure in general

Free format text: FINAL REJECTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: ADVISORY ACTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION