Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/22—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
  • C07K16/3076—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties
  • C07K16/3084—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties against tumour-associated gangliosides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/24—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/32—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
• • 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
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
• • 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
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/005—Glycopeptides, glycoproteins
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
  • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
  • C07K2317/41—Glycosylation, sialylation, or fucosylation
• • 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
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
  • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
• • 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
  • C12N2500/00—Specific components of cell culture medium
  • C12N2500/30—Organic components
  • C12N2500/34—Sugars

Definitions

• the conditions used for modulating the fucosylated glycan content for a particular recombinant protein should be selected so as not to affect or significantly alter the amount or level of any other glycan profile.
• a combination of conditions for obtaining a specific fucosylation profile should have no significant impact on titer and/or productivity of the process.
• the population of Hul4.18K322A monoclonal antibodies is provided in a pharmaceutical composition, wherein said antibodies are in admixture with a physiologically acceptable diluent, carrier, or excipient.
• the invention also provides a mammalian host cell harboring a nucleic acid encoding Hul4.18K322A monoclonal antibody and selected for producing substantially afucosylated Hul4.18K322A monoclonal antibody (e.g., at least 55% afucosylation) and a mammalian host cell deposited under American Type Culture Collection accession number XXXX on February 13, 2019.
• the cells belonging to mammals are preferably myeloma cells, ovarian cells, renal cells, blood cells, uterine cells, connective tissue cells, mammary cells, embryonic retinoblastoma cells, or cells derived therefrom, and more preferably cells selected from myeloma cells, myeloma cell-derived cells, ovarian cells, and ovarian cell-derived cells.
• hamster cell lines such as CHO-K1 (ATCC No. CCL-61) , CHO/dhfr- (ATCC No. CRL-9096) , CHO/DG44 (Urlaub & Chasin (1980) Proc . Natl. Acad. Sci. USA 77:4216) and BHK21 (ATCC No. CRL-10); dog cell lines such as MDCK (ATCC No. CCL-34), and the like.
• G-MEM Essential Medium
• PF CHO SAFC Biosciences
• POWERCHOTM 2 Lidza
• ZAP-CHO Invitria
• ProCHOTM Liscove's Modified Dulbecco's Medium
• the basal medium is modified to remove certain non-nutritional components found in a standard or conventional basal medium, such as various inorganic and organic buffers, surfactant (s) , and sodium chloride. Removing such components from basal cell medium allows an increased concentration of the remaining nutritional components, and may improve overall cell growth and protein expression.
• a modified basal medium excludes any, if not all, of the following ingredients: sodium bicarbonate, a buffer, mono-basic sodium phosphate, di -basic sodium phosphate, and a surfactant. See, US 9,234,032.
• the fed-batch process may also include the addition of cysteine or a cysteine derivative, such as N-acetyl cysteine, to the basal medium.
• cysteine is included in the cell culture medium in an amount of about 0.1 to 10 mM, or more preferably about 1 to 7 mM cysteine.
• an energy source is also added to the cell culture medium of the invention.
• the energy source is a monosaccharide.
• monosaccharides which may be used in the cell culture medium include glucose (e.gr., D- glucose) , maltose, mannose, galactose and fructose.
• glucose is added to the cell culture medium at a final concentration ranging from 0.5-4.0 g/L.
• glucose is added to the cell culture medium at a final concentration of no greater than 4.0 g/L.
• glucose is added to the cell culture medium at a final concentration of about 0.5 to 1.5 g/L and most preferably 1.0 g/L.
• the cell culture medium of the invention may further include glutathione. In one embodiment, 0.4 mg/L to 1.65 mg/L glutathione is added to the cell culture medium.
• the cell culture medium includes a recombinant growth factor such as insulin or a recombinant analog, IGF-1, or a combination of insulin and IGF-1.
• a recombinant growth factor such as insulin or a recombinant analog, IGF-1, or a combination of insulin and IGF-1.
• 4 mg/L to 13 mg/L insulin or a recombinant analog is added.
• 25 ng/L to 150 ng/L IGF-1 is added.
• 50 ng/L to 100 ng/L IGF-1 is added.
• 25 ng/L to 150 ng/L IGF-1 is supplemented to the insulin.
• 50 ng/L to 100 ng/L IGF-1 is supplemented to the insulin.
• a Hul4.18K322A antibody produced in the accordance with the present method has enhanced antibody- dependent cell -mediated cytotoxicity (ADCC) activity.
• An antibody having an "enhanced ADCC activity" refers to an antibody that is more effective at mediating ADCC in vitro or in vivo compared to the parent antibody, wherein the antibody and the parent antibody differ in at least one structural aspect, and when the amounts of such antibody and parent antibody used in the assay are essentially the same.
• the antibody and the parent antibody have the same amino acid sequence, but the antibody is afucosylated while the parent antibody is fucosylated.
• ADCC activity will be determined using the in vitro ADCC assay, but other assays or methods for determining ADCC activity, e.gr., in an animal model etc., are contemplated.
• the antibody is purified such that no polypeptide bands corresponding to other polypeptides are detectable upon analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) .
• SDS-PAGE SDS-polyacrylamide gel electrophoresis
• multiple bands corresponding to the antibody can be visualized by SDS-PAGE, due to differential glycosylation, differential post-translational processing, and the like.
• the antibody of the invention is purified to substantial homogeneity, as indicated by a single polypeptide band upon analysis by SDS-PAGE.
• the polypeptide band can be visualized by silver staining, Coomassie blue staining, or (if the polypeptide is radiolabeled) by autoradiography.
• Electroporation is used to introduce the DNA encoding the Hul4.18K322A antibody described above into Chinese hamster ovary (CHO) cells or rat hybridoma cells YB2/0.
• CHO Chinese hamster ovary
• rat hybridoma cells YB2/0 Chinese hamster ovary
• electroporation cells are grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine and penicillin/streptomycin. About 5xl0 e cells are washed once with PBS and resuspended in 0.5 ml PBS .
• Stably transfected clones are selected by their growth in the presence of methotrexate (MTX) .
• MTX methotrexate
• parent clone #108-334 was identified from subcloning by limiting dilutions . Subcloning was carried out by plating cells at 8, 4, 2, 1, 0.5 and 0.25 cells/well in 96-well plates containing DMEM supplemented with 10% FBS, 1 mM sodium pyruvate, 2 mM glutamine, penicillin/streptomycin, and 50 nM MTX. When subclones appeared on the plates containing 0.25 cell/well, 0.5 cell/well and 1 cell/well, the plates containing at least 2 cells/well were discarded.
• Hul4.18K322A producing cells were maintained in HSFM supplemented with 6 mM GlutamaxTM, 2 g/L soytone and 2 g/L phytone hydrolysate and either 52 nM MTX for parent clone #108-334 or 1000 nM MTX for production clone #134.
• the first step in the production of Hul4.18K322A was a standard inoculum seed train to generate cells to inoculate the production reactor. A vial from the master cell bank was thawed and cells were inoculated at a viable cell density of 0.2-0.3 x 10 s cells/mL in suspension.
• hul4.18K322A titers of 130 mg/L were achieved with an afucosylation percentage (%AF) of 54% for clone #108-334.
• Reactors were clarified via depth filtration followed by sterile (0.2 mm) filtration and used for downstream processing.
• a PROMEGA ADCC reporter bioassay is used to assess ADCC activity.
• a diluted stock of antibody is prepared by diluting the antibody to 1:1000 using the ADCC Assay buffer.
• the diluted stock is further diluted to 1 pg/mL using the ADCC Assay buffer.
• Effector Cells Effector cells are thawed in a 37°C water bath for 2-3 minutes, followed by pipetting 630 pL of the cells into 3.6 mL of pre-warmed ADCC Assay buffer. The cells are mixed by gently pipetting 1-2 times in the assay buffer. Effector cells are transferred to sterile reagent reservoir and 25 pL are subsequently pipetted into wells containing target cells ⁇ antibody. The plates are returned the tissue culture incubator and incubated for six hours.
• Luciferase assay buffer is thawed at room temperature and used in the reconstitution of luciferase reagent powder. After the six-hour incubation, plates are removed from the tissue culture incubator and placed at room temperature for fifteen minutes. To each of well is added 75 pL of the room temperature, reconstituted Bio-Glow reagent. Cells are incubated with the Bio-Glow reagent at room temperature for 15 minutes. The plates are incubated in the dark.

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