WO2013089496A1 - Anticorps monoclonal neutralisant à large spectre ayant un potentiel élevé issu de patients infectés par h1n1, et composition contenant celui-ci pour le traitement du virus - Google Patents

Anticorps monoclonal neutralisant à large spectre ayant un potentiel élevé issu de patients infectés par h1n1, et composition contenant celui-ci pour le traitement du virus Download PDF

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WO2013089496A1
WO2013089496A1 PCT/KR2012/010929 KR2012010929W WO2013089496A1 WO 2013089496 A1 WO2013089496 A1 WO 2013089496A1 KR 2012010929 W KR2012010929 W KR 2012010929W WO 2013089496 A1 WO2013089496 A1 WO 2013089496A1
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antibody
virus
present
seq
monoclonal antibody
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Korean (ko)
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김호언
배소현
이은숙
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(주)에이프로젠
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • 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
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to a very potential broad-spectrum neutralizing monoclonal antibody derived from H1N1-infected patients.
  • the present invention relates to an antiviral therapeutic composition comprising the monoclonal antibody.
  • Influenza viruses are classified into types A, B, and C, depending on the composition of the proteins.
  • Type A and B can be touched by humans.
  • Type A has a much faster rate of transition than Type B and Type C.
  • Type A is mostly found in wildbirds, sometimes spreading to poultry (chicken, etc.) or humans.
  • Influenza viruses have hemagglutinin (H) and neuraminidase (N) in their envelope lipid layers.
  • H1, H2, H3, ... and N1, N2, N3, There are several variants, depending on the type of variant (H1, H2, H3, ... and N1, N2, N3, ).
  • Representative examples include H1N1, also known as swine flu or swine flu, and others such as H5N1, H1N2, H9N2, H7N2, H7N3, and H10N7.
  • H1N1 caused a flu pandemic called the Spanish flu, and between 1918 and 1919 killed between 50 and 100 million people.
  • the low pathogenic H1N1 is now widely distributed around the world, accounting for nearly half of human infections in 2006.
  • Influenza A virus subtype H5NA is a subtype of influenza A, which causes highly pathogenic avian influenza. It can also cause transmission to humans and other animals, and currently no vaccines have been developed, and treatments such as Tamiflu and Relenza are being used.
  • Neutralizing antibodies are antibodies that protect cells from antigens or infectious agents by inhibiting or neutralizing biological activity.
  • Antibodies are produced in non-human mammals using HN protein sequences that contain epitopes that do not show an immune response in humans upon infection or natural exposure to the environment, neutralizing HN infectivity, and easily killing infected CD4 lymphocytes. In addition, it may be administered to inactivate an essential step in the life cycle of HN.
  • 'neutralization site' refers to such a portion of an HN, in particular HN protein, comprising an amino acid segment that defines one or more epitopes that are reactive with an antibody capable of binding to other antibodies of the invention or individually neutralizing HN infection. Suitable assays for investigating neutralization are also known and may include assays to measure reduction of HN infection, cochlear inhibition test and virion-receptor binding test in the T-cell system.
  • the term 'inert site' refers to a segment of an HN protein comprising one or more epitopes that, when combined with or individually reacted with an antibody, functionally inactivate important events in the HN life cycle.
  • Suitable assays for investigating the destruction rate of antibody-mediated HN infected lymphocytes are known and may include antibody dependent cell mediated cytotoxicity, complement mediated lysis and natural killer (NK) assays.
  • Suitable assays for determining antibody-mediated inactivation of essential stages of the HN life cycle can be determined by determining inactivation of reverse transcriptase, measuring polymerase and protease activity, or by exposing viral RNA to ribonuclease digestion.
  • Assays to assess antibody mediated complement dependency changes in capsid permeability.
  • immunochemical assays such as immunofluorescence, immunoblot, enzyme-linked immunoassay and radioimmunoassay, neutralizing activity can be compared with antibody activity.
  • An object of the present invention is to provide an antibody exhibiting neutralization over a wide range.
  • Another object of the present invention is to provide a pharmaceutical composition comprising an antibody exhibiting neutralization over a wide range.
  • the first aspect of the present invention is an antibody comprising SEQ ID NO: 1 in a heavy chain variable portion.
  • the basic structure of the antibody is a Y-shaped protein, and has two specific structures that can bind antigens to the upper two of the Y. This specific structure can have an unimaginably large number of genes at the DNA level that occurs in immune cells, and thus can respond to a variety of antigens.
  • the basic unit of the antibody is a Y-shaped monomer molecule composed of two light chains and two heavy chains each disulfide-linked, and monomers of 1-5 form a single antibody. There are five types of heavy chains ( ⁇ , ⁇ , ⁇ , ⁇ , ⁇ ), and the heavy chain determines the type of antibody. ⁇ and ⁇ are composed of 450 amino acids and ⁇ and ⁇ are composed of 550 amino acids. There are two regions in the heavy chain: variable regions and constant regions.
  • variable region depends on the B cell (plasma cell) from which the antibody is made, and all of the variable regions of the antibody made in the same cell are identical.
  • the primary structure is numerous in the same individual to form antigen binding sites of antibody molecules.
  • the constant regions are identical only when the antibodies are the same type. ⁇ , ⁇ , and ⁇ have three constant regions and bend regions, while ⁇ and ⁇ have four constant regions.
  • the variable regions are linked together by amino acids.
  • the light chain consists of a constant region and a variable region in succession.
  • the Y-shaped antibody monomer is composed of two heavy chains and two light chains, and has 6-8 constant regions and 4 variable regions.
  • the fragments of the antibody (branch of Y) are called Fab fragments (Fab fragment fragments), each having one constant region and one variable region, and the N-terminus binds to the antigen.
  • Two variable regions are sites that bind to specific antigens.
  • the gist of the present invention can be achieved by partially deleting, adding to, or substituting the amino acid sequence of SEQ ID NO: 1 using the gist of the present invention. have. Accordingly, the scope of the present invention also includes amino acid sequences in which the amino acid of SEQ ID NO: 1 is partially substituted, added, or deleted within the scope of achieving the object of the present invention. Substitutions of amino acids are preferably conservative substitutions.
  • amino acids present in nature are as follows; Aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn ) And sulfur-containing amino acids (Cys, Met). Deletion of amino acids is preferably located at portions not directly involved in the activity of the antibody.
  • the second aspect of the present invention is a hexane sequence encoding the amino acid sequence of SEQ ID NO: 1. Due to the degeneracy of the genetic code, even the hexane sequence of various forms can encode the amino acid sequence of SEQ ID NO: 1 identically.
  • the scope of the present invention includes all hexane sequences encoding the amino acid sequence of SEQ ID NO: 1.
  • a third form of the invention is a recombinant vector comprising a hexane sequence encoding SEQ ID NO: 1.
  • vector refers to a nucleic acid molecule capable of binding and transferring another nucleic acid thereto.
  • expression vector includes plasmids, cosmids or phages capable of synthesizing proteins encoded by each recombinant gene carried by the vector.
  • Preferred vectors are vectors capable of self-replicating and expressing the nucleic acid to which they are bound.
  • a fourth form of the invention is a host cell transformed with a recombinant vector comprising a hexane sequence encoding SEQ ID NO: 1.
  • the term 'transformation' used in the present invention means that foreign DNA or RNA is absorbed into a cell and the genotype of the cell is changed.
  • Suitable transformed cells include, but are not limited to, prokaryotes, fungi, plants, animal cells, and the like. Most preferably, E. coli is used.
  • a fifth aspect of the present invention is an antibody comprising SEQ ID NO: 2 in a heavy chain variable portion.
  • the gist of the present invention can be achieved by partially deleting, adding to, or substituting the amino acid sequence of SEQ ID NO: 2 using the gist of the present invention. have. Accordingly, the scope of the present invention also includes amino acid sequences in which the amino acid of SEQ ID NO: 2 is partially substituted, added, or deleted within the scope of achieving the object of the present invention. Substitutions of amino acids are preferably conservative substitutions.
  • amino acids present in nature are as follows; Aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn ) And sulfur-containing amino acids (Cys, Met). Deletion of amino acids is preferably located at portions not directly involved in the activity of the antibody.
  • a sixth aspect of the present invention is a hexane sequence encoding the amino acid sequence of SEQ ID NO: 2. Because of the degeneracy of the genetic code, even the hexane sequence of various forms can encode the amino acid sequence of SEQ ID NO. The scope of the present invention includes all hexane sequences encoding the amino acid sequence of SEQ ID NO: 2.
  • a seventh form of the invention is a recombinant vector comprising a hexane sequence encoding SEQ ID NO: 2.
  • vector refers to a nucleic acid molecule capable of binding and transferring another nucleic acid thereto.
  • expression vector includes plasmids, cosmids or phages capable of synthesizing proteins encoded by each recombinant gene carried by the vector.
  • Preferred vectors are vectors capable of self-replicating and expressing the nucleic acid to which they are bound.
  • An eighth form of the invention is a host cell transformed with a recombinant vector comprising a hexane sequence encoding SEQ ID NO: 2.
  • the term 'transformation' used in the present invention means that foreign DNA or RNA is absorbed into a cell and the genotype of the cell is changed.
  • Suitable transformed cells include, but are not limited to, prokaryotes, fungi, plants, animal cells, and the like. Most preferably, E. coli is used.
  • a ninth aspect of the present invention is an antibody comprising SEQ ID NO: 1 in the heavy chain variable portion and SEQ ID NO: 2 in the light chain variable portion.
  • a tenth aspect of the present invention is a pharmaceutical composition for virus neutralization comprising the antibody comprising SEQ ID NO: 1 or SEQ ID NO: 2, and SEQ ID NO: 1 and 2.
  • the virus may be, but is not limited to, an influenza virus.
  • the virus may be an influenza virus having hemagglutinin of H1, H2, H5, H9. Even more preferably the virus may be H1N1 influenza virus or H5N1 influenza virus.
  • compositions of the present invention may be formulated in the form of a solid, liquid or aerosol to suit the route intended for administration.
  • solid compositions include tablets, creams, and implantable dosage units. Tablets can be administered orally.
  • Therapeutic creams can be used topically.
  • Implantable dosage units may be used locally, for example, at specific sites, and may be implanted, for example subcutaneously, to systemically release the pharmaceutical composition of the invention.
  • liquid compositions include subcutaneous, intravenous, intraarterial, topical and intraocular administration.
  • aerosol compositions include inhalable formulations for administration through the lungs.
  • compositions can be administered by standard routes.
  • the compositions can be administered by topical, oral, rectal, intravaginal, nasal or parenteral routes (eg, intravenous, subcutaneous, intramuscular).
  • the composition may comprise a sustained release matrix, such as a biodegradable polymer, eg, a polymer implanted at a target site. Biodegradable polymers and other polymers may also be implanted to allow for systematic delivery. Dosage methods include single dose administration, repeated dose administration at predetermined time intervals, and continuous administration at predetermined time intervals.
  • sustained release matrices are matrices made of materials, primarily polymers degradable by enzymatic or acid group hydrolysis or dissolution. Once inserted into the body, the matrix is activated by enzymes and body fluids.
  • the sustained release matrix is liposomes, polylactide acid, polyglycolide (polymer of glycolic acid), polylactide co-glycolide (lactic acid and glycolic acid) Polymers, copolymers of lactic acid and glycolic acid, polyanhydrides, poly (ortho) esters, polypeptides, hyaluronic acid, collagen , Chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, phenylalanine ), Amino acids such as tyrosine, isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone Biocompatible materials
  • composition depends on the therapeutic situation, the particular composition use and other clinical factors such as body weight and patient's condition, dosage form.
  • the composition can be administered in combination with the course of treatment of the disease and with other compositions.
  • routes of administration include parenteral or oral administration, such as intravenous, blood, subcutaneous, inhalation, topical, mucosal and rectal.
  • Solutions or suspensions used for oral administration such as blood or subcutaneous administration include solvents for injection, saline solutions, fixed oils, aseptic diluents such as polyethylene glycol, glycerin, propylene glycol or other synthetic solvents, such as benzine alcohol or methyl parabens.
  • Bacterial agents; Antioxidants such as ascorbic acid or sodium sulfite; Chelating agents such as ethylenediaminetetraacetic acid; Buffers such as acetate, citrate or phosphate and extender modifiers such as sodium chloride or dextrose may be included.
  • Suitable carriers for the preparation of suppositories are natural and hardened oils, waxes, fats, semi-liquid polyols and the like.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • Parenteral formulations may be sealed with ampoules, disposable syringes, multiple dose vials made of glass or plastic.
  • Suitable disease therapeutic agents for injection include sterile aqueous solutions or dispersions and sterile powders for the instant preparation of sterile injections or dispersions.
  • Suitable carriers for intravenous administration include physiological saline, bacteriostatic water, CremophorELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
  • the disease treatment agent must be sterile and must be fluid to the extent that it can be easily injected. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be, for example, a solvent or spray medium containing water, ethanol, polyols (glycerol, propylene glycol and liquid polyreylene glycol, etc.), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of by-products, and by using surfactants.
  • Microbial action can be prevented by various antibacterial and antibacterial agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. It is preferable that sugars, polyhydric alcohols such as mannitol, sorbitol, and isotonic agents such as sodium chloride are included in the disease treatment agent. Prolonged absorption of the injectable composition can be achieved by, for example, incorporating delayed absorption agents such as aluminum monostearate and gelatin in the treatment of disease.
  • Sterile injectable solutions can be prepared by incorporating the required active ingredient in one or a combination of the ingredients described above in a suitable solvent and, if necessary, by filter sterilization.
  • dispersions can be prepared by incorporating the active ingredient in a sterile vehicle which may contain the basic dispersion medium and the required other ingredients from those described above.
  • sterile powders for the preparation of sterile injectable solutions the preferred methods of preparation are vacuum drying and lyophilization, by which methods powders of the desired ingredient can be obtained from the active ingredient and the previously sterilized filtration solution.
  • suitable carriers for the preparation of injectable solutions include water, alcohols, polyols, glycerin, vegetable oils and the like.
  • Oral compositions generally include an inert diluent or an edible carrier.
  • the active ingredient is incorporated into excipients and used in capsules in the form of tablets, troches, or gelatin. Oral use may also be prepared using a fluid carrier for use as an oral lavage fluid.
  • suitable carriers for soft gelatin capsules are vegetable oils, waxes, fats, semi-solid and liquid polyols and the like.
  • suitable carriers for the preparation of solutions and syrups are water, polyols, saccharose, invert sugar, glucose and the like.
  • compositions may also contain preservatives, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, salts for controlling osmotic pressure, buffers, coatings or antioxidants.
  • Tablets, pills, capsules, trucks and the like may contain any or a mixture of similar properties of the following components: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; Excipients such as starch or lactose, disintegrants such as alginic acid, Primogel TM or corn starch; Lubricants such as magnesium stearate or Sterotes TM; Gildants, such as silicon dioxide in the colloidal state; Sweetening agents such as sucrose or saccharin; Or flavoring agents such as peppermint, methyl salicylate, or orange flavor. Oral or parenteral disease therapeutic agents are advantageously formulated in dosage unit form to facilitate uniformity under dose administration.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • Excipients such as starch or lactose, disintegrants such as alginic acid, Primogel TM or corn starch
  • Lubricants such as magnesium stearate or Sterotes TM
  • Gildants
  • the miRNAs may be formulated as a medicament in a conventional manner using one or more pharmaceutically acceptable carriers. Lactose, corn starch or derivatives thereof, talc, stearic acid or its salts can be used, for example, as carriers for tablets and hard gelatin capsules.
  • the dosage for the treatment or prophylaxis of the virus can vary widely and can, of course, be adjusted to the individual requirements in each particular case.
  • Antibodies according to the invention can be neutralized in a broad spectrum to all influenza viruses comprising hemagglutinin of H1, H2, H5 and H9.
  • the antibody according to the present invention has an excellent binding affinity for the antigen.
  • FIG. 1 shows the amino acid sequence of the variable domains in the heavy chain and the light chain of antibody 4D9 according to the present invention.
  • Figure 2 shows a chart comparing the antigen binding affinity through ELISA of antibodies 4D9 and F10 according to the present invention. It is shown here that antibody 4D9 according to the present invention binds to H1, H2, H5 and H9 with a higher affinity than F10 (nAb concentration. ⁇ 100 ng / ml).
  • Figure 3 shows the antigen binding spectrum of antibody 4D9 according to the present invention through a flow cytometer. Similar to antibody F10, except for antibodies 4D9 d H3 and H7 according to the present invention, H1, H2, H5 and H9 bind. (Remicade: negative control)
  • FIG. 4 shows a competitive ELISA between antibody F10 and antibody 4D9 according to the present invention. This indicates that the two antibodies share the same antigenic determining site.
  • FIG. 5 shows that antibody 4D9 according to the present invention inhibits H5-mediated cell fusion.
  • Antibody 4D9 was obtained from an immune library prepared from the blood of H1N1 influenza patients during the recovery phase.
  • Bacteria: helper phage 1: 20 was inoculated to helper phage and stood at 37 °C for 30 minutes and then shaken for 200rpm, 30 minutes.
  • Cell pellets obtained by centrifuging the culture solution at 6000 rpm for 15 minutes were resuspended in 2 L 2xYT + 70 ug / ml kanamycin, 100 ug / ml ampicillin, 5 mM MgCl 2 medium, and cultured overnight at 220 rpm and 30 ° C. The next day, the culture solution was centrifuged at 3000 rpm for 30 minutes, and only the supernatant was taken up, and 4% PEG 8000 and 3% NaCl were added and completely dissolved. This was allowed to stand for more than 1 hour on ice and centrifuged at 8000 rpm for 40 minutes to remove the supernatant.
  • the pellets were dried for 10 minutes and then resuspensioned in 1 ⁇ PBS. Transfer to 1.5 ml tube and centrifuged for 10 minutes at 12,000 rpm. Only the supernatant was taken and divided into 500 ul and stored at -70 ° C.
  • For titration of phage grow ER2738 cells with OD 0.5 ⁇ 0.7. Serial dilution of phage solution to 1/100 is carried out in 100 ul of ER2738 cells 10 ul each. This was incubated at 37 °C for 15 minutes, spread on a 2xYT + ampicillin plate and incubated overnight at 37 °C. This process was repeated in pairs with the panning process below.
  • a phage was displayed to display the antibody using the cell stock that emerged after each panning. Inoculate ER2738 cells in 25 ml 2xYT medium and grow at 220 rpm and 37 ° C until the OD is about 0.5. The day before panning, two immunosorb tubes were coated with 5 ug / ml antigen (purified H5N1-HA VN04) and one immunosorb tube was coated with 5 ug / ml BSA (4, overnight). The coating solution was discarded and blocked with 4% skim milk diluted in PBS for 37 and 2 hours.
  • 2xYT + ampicillin + glucose medium was added to the deep plate at 200 ul / well, and 96 colony per plate was inoculated therein, followed by incubation at 37 ° C and 600 rpm for 6 hours. At this time, the hand towel was sterilized and put on the lid to prevent cross contamination by water vapor. After incubation, 20 ul of the culture solution was inoculated into a new deep plate containing 180 ul / well of 2xYT + ampicillin medium and incubated at 37 ° C and 600 rpm for 1 hour. The plate after inoculation was stored at -70 °C with 15% glycerol and used as a master plate.
  • helper phage of 3 ul / well was added and allowed to stand for 30 minutes at 37 °C and incubated for 30 minutes at 600 rpm. After incubation, 2 ul / well of kanamycin was added and incubated overnight at 30 ° C. The next day, the plate was centrifuged at 2000 rpm for 10 minutes, the supernatant was taken at 150 ul each, mixed in a plate containing 150% of 4% skim milk, and then reacted at room temperature for 30 minutes to use for ELISA. One day, the ELISA plate was coated with 200 ng / well of antigen (purified H5N1-HA VN04) and BSA (4, overnight).
  • the coating solution was discarded and reacted for 2 hours at 37 ° C with 4% skim milk diluted in PBS.
  • the prepared phage + skim milk solution was put into 100 ul / well and reacted at 37 ° C. for 1 hour.
  • anti-M13-HRP was diluted 1: 1000 in TBST and put into 100 ul / well, followed by reaction at 37 ° C for 1 hour.
  • OPD OPD
  • the reaction was stopped with 50 ul of 1M H 2 SO 4.
  • the absorbance was measured at 490 nm using an ELISA reader.
  • the variable sequence of the antibody was obtained by DNA sequencing of 4D9 clone with the highest ELISA.
  • Figure 2 shows a chart comparing the antigen binding affinity through ELISA of antibodies 4D9 and F10 according to the present invention. It is shown here that antibody 4D9 according to the present invention binds to H1, H2, H5 and H9 with a higher affinity than F10 (nAb concentration. ⁇ 100 ng / ml).
  • an ELISA plate was coated with HA at a concentration of 100 ng / ml (4, overnight). After washing three times with 0.05% TBST the next day, 20% BSA diluted in PBS was treated with 200 ul / well for 1 hour at room temperature.
  • Figure 3 shows the antigen binding spectrum of antibody 4D9 according to the present invention through a flow cytometer. Similar to antibody F10, except for antibodies 4D9 d H3 and H7 according to the present invention, H1, H2, H5 and H9 bind. (Remicade: negative control)
  • pJK2b-HA was transfected into 293T cells to see how 4D9 binds to HA bound cells.
  • 293T cells were inoculated into 6 well plates and incubated in 37 ° C., 5% CO 2 incubator using DMEM containing 10% FBS for 2 days.
  • pJK2b-HA 4 and Lipofectamin 2000 (invitrogen, cat # 11668) 10 are diluted in Opti-MEM I (invitrogen, cat # 31985) 250, respectively, and the expression vector and lipofectamine are prevented from forming. Mix 2000 and let the mixture stand at room temperature for 15 minutes.
  • FIG. 4 shows a competitive ELISA between antibody F10 and antibody 4D9 according to the present invention.
  • a compitition ELISA was performed to confirm whether 4D9 and F10 bind to the same epitope.
  • the ELISA plate was coated with 200 ng / well of HA1 and HA5 for 4, overnight. The next day the coating solution was discarded and blocked with 4% skim milk diluted in PBS for 2 hours at 37 ° C.
  • FIG. 5 Shows that antibody 4D9 according to the present invention inhibits H5-mediated cell fusion.
  • a cell fusion inhibition assay to determine how 4D9 inhibits cell fusion induced by HA.
  • the mixture was allowed to stand for 15 minutes at room temperature by mixing the expression vector and Lipofectamine 2000 to prevent bubbles. After incubation for 5 hours, the cells were replaced with DMEM + 10% FBS medium for 2 days. After incubation, the reaction was replaced with serum-free DMEM medium containing 4D9 for 1 hour. After washing with PBS, acid medium of pH4.8 containing 0.1M citric acid was treated for 15 minutes, washed with PBS again, and then incubated with DMEM + 10% FBS medium for 3 hours. After incubation, the fusion pattern was observed under a microscope. After HA5 transfection in HeLa cells, cell fusion was observed throughout the plate and 50-60% inhibition was observed in 4D9 treatment.

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Abstract

La présente invention concerne un anticorps monoclonal neutralisant à large spectre ayant un potentiel élevé issu à partir de patients infectés par H1N1, et une composition contenant celui-ci pour le traitement du virus. Plus particulièrement, la présente invention concerne un anticorps monoclonal comprenant SEQ ID N° 1 et 2, et une composition comprenant l'anticorps monoclonal comme principe actif pour la prévention et le traitement du virus.
PCT/KR2012/010929 2011-12-15 2012-12-14 Anticorps monoclonal neutralisant à large spectre ayant un potentiel élevé issu de patients infectés par h1n1, et composition contenant celui-ci pour le traitement du virus WO2013089496A1 (fr)

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JP2019530641A (ja) * 2016-07-19 2019-10-24 アイベントラス・インコーポレイテッドＩｂｅｎｔｒｕｓ，Ｉｎｃ． 二重特異性タンパク質およびその製造方法
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US11230593B2 (en) 2019-03-25 2022-01-25 Visterra, Inc. Compositions and methods for treating and preventing influenza

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