WO2012012271A2 - Nouveaux polypeptides glycosylés - Google Patents

Nouveaux polypeptides glycosylés Download PDF

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Publication number
WO2012012271A2
WO2012012271A2 PCT/US2011/044081 US2011044081W WO2012012271A2 WO 2012012271 A2 WO2012012271 A2 WO 2012012271A2 US 2011044081 W US2011044081 W US 2011044081W WO 2012012271 A2 WO2012012271 A2 WO 2012012271A2
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neu5gc
composition
human
cells
sialic acid
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PCT/US2011/044081
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WO2012012271A3 (fr
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Ajit Varki
Darius Ghaderi
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Sialix, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig
    • 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
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation

Definitions

  • glycosylated polypeptide compositions relate to glycosylated polypeptide compositions and their uses.
  • the glycosylated polypeptides of the invention include, for example, polypeptides with reduced Neu5Gc.
  • Glycosylated polypeptides are often produced in mammalian expression systems.
  • Non-human cell lines such as rodent cell lines, used to produce glycosylated polypeptides generate N-glycans similar to those of humans, with a few exceptions.
  • CMAH CMP-N-acetylneuraminic acid hydroxylase
  • CMP-Neu5Ac CMP-Neu5Ac
  • polypeptides produced from human cells generally lack Neu5Gc.
  • Neu5Gc can be taken up from animal products present in the culture medium and then metabolically incorporated into secreted glycoproteins by human cells .
  • human cells cultured with animal-derived supplements will secrete glycoproteins bearing Neu5Gc.
  • Neu5Gc is not part of the normal glycosylation pattern of human proteins and humans have been observed to generate high levels of circulating anti-Neu5Gc antibodies to the Neu5Gc-contaminated glycoproteins.
  • Neu5Gc-carrying agents may load human tissues with this non-human sugar which, in combination with anti-Neu5Gc antibodies, mediate chronic inflammation and potentially facilitate progression of diseases such as cancer 3 and atherosclerosis 4 .
  • chronic use of Neu5Gc-bearing therapeutics may increase the risk of developing such diseases.
  • Neu5Gc-bearing therapeutics can be cleared faster from the bloodstream due to anti-Neu5Gc antibodies and therefore have lowered efficacy relative to the same therapeutic with a lowered Neu5Gc content.
  • compositions of the invention include recombinant human glycosylated polypeptides or non- human animal glycosylated polypeptides in which Neu5Gc is substantially reduced or eliminated.
  • compositions include, but are not limited to, a polypeptide such as a monoclonal antibody, Fc-fusion protein, hormone, cytokine, clotting factor, enzyme inhibitor, enzyme and antiserum.
  • a polypeptide such as a monoclonal antibody, Fc-fusion protein, hormone, cytokine, clotting factor, enzyme inhibitor, enzyme and antiserum.
  • the monoclonal antibody is Tocilizumab, Bevacizumab, Alemtuzumab, Trastuzumab, Adalimumab, Rituximab, Golimumab, Ustekinumab, Panitumumab, Omalizumab, Ibritumomab tiuxetan, Tositumomab-1131, Eculizumab, Canakinumab, Gemtuzumab ozogamicin, Ofatumumab, Palivizumab, Natalizumab, Cetuximab, Infliximab, Abciximab, Basiliximab, Daclizumab, Certolizumab pegol, or Ranibizumab.
  • the Fc-fusion protein is Alefacept, Rilonacept, Etanercept, Abatacept, or Romiplostim.
  • the hormone is Follitropin beta, Follitropin alfa, Luteinizing hormone, Osteogenic Protein- 1 (BMP-7), Choriogonadotropin alpha, Thyrotropin alfa, Somatropin, keratinocyte growth factor, Calcitonin, or Platelet-derived growth factor (PDGF).
  • BMP-7 Osteogenic Protein- 1
  • Choriogonadotropin alpha Choriogonadotropin alpha
  • Thyrotropin alfa Somatropin
  • keratinocyte growth factor keratinocyte growth factor
  • Calcitonin Calcitonin
  • PDGF Platelet-derived growth factor
  • the cytokine is Darbepoetin alfa, Interferon beta- la, Epoetin beta, Epoetin alfa, Interferon beta- la, Interferon gamma- lb, Interferon alfacon-1, Interferon alfa-2b, interleukin-1 receptor antagonist (IL-IRa), Pegfilgrastim, Des-Pro Interleukin-11, G-CSF , IL- 2/diphtheria toxin fusion protein, Peginterferon alfa-2a, Aldesleukin (IL-2), or Interferon alfa-2a.
  • IL-IRa interleukin-1 receptor antagonist
  • the clotting factor is Coagulation factor VIII, Coagulation Factor Vila, Antihemophilic factor, Coagulation Factor IX, Antihemophilic Factor, or Drotrecogin alfa (Activated Protein C).
  • the enzyme is Alteplase, Laronidase, Imiglucerase, agalsidase- ⁇ , yaluronidase, Alglucosidase alfa, N-acetylgalactosamine 4-sulfatase, Human DNase, Tenecteplase, Idursulfase, Collagenase, or Rasburicase.
  • the glycosylated polypeptide is recombinant (i.e., produced by recombinant protein expression methods). In other embodiments the glycosylated polypeptide is mammalian, including human and/or non-human polypeptides.
  • the composition (e.g., a glycosylated polypeptide) is derived from a cell line fed Neu5Ac, e.g. cultured in media containing Neu5Ac.
  • the cell line is derived from a non-human animal.
  • the cell line is derived from a human.
  • the culture cells are hybridomas, Chinese Hamster Ovary (CHO) cells, murine myeloma cells, murine CI 27 cells, Baby Hamster Kidney (BHK) cells, HT- 1080 or Human embryonic kidney cells (HEK293).
  • Polypeptide refers to a polymer in which the monomers are amino acids and are joined together through amide bonds, alternatively referred to as a polypeptide. Additionally, unnatural amino acids, for example, ⁇ -alanine, phenylglycine and homoarginine are also included. As used herein, “polypeptide” refers to both glycosylated and unglycosylated polypeptides. Also included are polypeptides that are incompletely glycosylated by a system that expresses the peptide. For a general review, see, Spatola, A. F., in CHEMISTRY AND BIOCHEMISTRY OF AMINO ACIDS, PEPTIDES AND PROTEINS, B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983). The term polypeptide includes molecules that are commonly referred to as peptides or proteins.
  • An "isolated” or “purified” glycosylated polypeptide or biologically-active portion thereof is substantially free of cellular material or other contaminating polypeptides from the cell or tissue source from which the glycosylated polypeptide is derived.
  • glycosylated polypeptide means a polypeptide having at least one carbohydrate moiety covalently linked thereto. It is understood that a glycosylated polypeptide may be a “therapeutic glycosylated polypeptide.”
  • glycosylated polypeptide can be used interchangeably herein with the terms “glycopolypeptide,” “glycopeptide” and “glycoprotein.”
  • recombinant means derived from genetic engineering, e.g. a recombinant polypeptide isolated from a cell or organism wherein the nucleic acid coding for the polypeptide is from another organism, such a human gene coding for a human protein that is expressed in a Chinese Hamster Ovary (CHO) cell.
  • genetic engineering e.g. a recombinant polypeptide isolated from a cell or organism wherein the nucleic acid coding for the polypeptide is from another organism, such a human gene coding for a human protein that is expressed in a Chinese Hamster Ovary (CHO) cell.
  • expression includes but is not limited to one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and/or other modifications of the translation product, when required for proper expression and function.
  • compositions of the invention do not include human glycosylated polypeptides obtained directly from humans wherein the human glycosylated polypeptides substantially lack Neu5Gc, e.g. human glycosylated polypeptides obtained from human blood or tissue.
  • Neuro5Gc means N-glycolylneuraminic acid, which is converted from the sialic acid N-acetylneuraminic acid (Neu5Ac) by the activity of cytidine monophosphate hydroxylase (CMAH).
  • CMAH cytidine monophosphate hydroxylase
  • a "Neu5Gc competitor” is a compound, such as a sugar, that can compete with Neu5Gc in cell culture or non-human animal such that an alternate sugar can replace Neu5Gc on glycosylated polypeptides.
  • substantially reduced in Neu5Gc means that the human glycosylated polypeptide has a lower mol fraction of Neu5Gc than what is obtained when the human glycosylated polypeptide is produced in a non-human animal source, including, but not limited to (1) in a non-human animal cell line or (2) in a non-human animal cell line or a human cell line wherein the cell line is cultured in media that introduces Neu5Gc (such as by supplementing the media with fetal calf serum).
  • the glycosylated polypeptides can have a mol fraction of Neu5Gc of less than 2, of less than 1, of less than 0.5, of less than 0.2, of less than 0.1, of less than 0.05, of less than 0.02, of less than 0.01, of less than 0.005, of less than 0.002, or of less than 0.001.
  • the glycosylated polypeptide with substantially reduced Neu5Gc has a mol fraction of Neu5Gc of more than 0.01, more than 0.02, more than 0.05, more than 0.1, more than 0.5.
  • Neu5Gc content of a glycosylated polypeptide can be readily determined by those skilled in the art using immunodetection methods known in the art. 5
  • Figure 1 ELISA and Western-Blot Detection of Neu5Gc on Cet and Pan.
  • ELISA analysis of periodate-treated Cet and Pan (Panel C).
  • ELISA analysis of anti-Neu5Gc IgG-treated Cet and Pan (Panel D).
  • Western Blot of anti-Neu5Gc IgG-treated Cet and Pan (Panel E). Detection of immune complex formation with Cet or Pan in whole human serum (Panel F).
  • Figure 2 Effects of anti-Neu5Gc antibodies on the kinetics of Cet and Pan in mice with a human-like Neu5Gc-deficiency.
  • ELISA analysis of therapeutic antibody clearance kinetics in Cmah null mice injected with Cet and Pan (Panel A).
  • ELISA analysis of Neu5Gc specific antibodies in Cmah null mice were injected i.v. with Cet or Pan (Panel B).
  • ELISA analysis of direct binding of anti-Neu5Gc antibodies to Cet and Pan (Panel C).
  • FIG. 3 Reduction of Neu5Gc Contamination in Glycosylated Polypeptides of Human 294T Cells and CHO Cells.Neu5Gc and Neu5Ac content, relative to total sialic acid content, of ethanol soluble (Panel A) and ethanol precipitable proteins (Panel B) analyzed by HPLC in human 293T cells grown in the presence of 5 mM Neu5Gc for 3 days.
  • Neu5Gc content relative to total sialic acid content, of glycoproteins in CHO cells grown in the absence or presence of 5 mM Neu5Ac in secreted proteins (Panel C) and membrane-bound proteins (Panel D) analyzed by HPLC.
  • Suitable methods for substantially reducing Neu5Gc content in human glycosylated polypeptides isolated from tissue culture or non-human animals are disclosed in U.S Provisional Patent Application No. 61/095,414, titled "Elimination of a Contaminating Non-human Sialic Acid by Metabolic Competition," the entire contents of which including figures are incorporated by reference herein.
  • the human glycosylated polypeptides in which Neu5Gc is substantially reduced disclosed herein are produced by methods including the introduction of a Neu5Gc competitor to cells.
  • a Neu5Gc competitor can be used for metabolically competing out Neu5Gc, either as it enters the cells for the first time and/or when it recycles from breakdown of preexisting cellular molecules.
  • Neu5Gc competitors include, but are not limited to, N-acetylneuraminic acid (Neu5Ac), available from e.g. Sigma Aldrich (Munich, Germany), E.M.D.
  • methods for producing human glycosylated polypeptides in which Neu5Gc is substantially reduced include, but are not limited to, culturing a cell line with increased amounts of N-acetylneuraminic acid (Neu5 Ac) or its precursor N-acetylmannosamine (ManNAc), such as by adding such sugars as a medium supplement, for a sufficient period of time to substantially reduce Neu5Gc on glycosylated polypeptides present in a cell or cell line or product produced by a cell or cell line.
  • Neu5 Ac N-acetylneuraminic acid
  • ManNAc N-acetylmannosamine
  • Cells in culture can be supplemented with Neu5Gc competitor with at a concentration of at least 100 ⁇ , at least 200 ⁇ , at least 500 ⁇ , at least 1 mM, at least 2 mM, at least 5 mM, at least 10 mM, at least 20 mM, at least 50 mM, at least 100 mM, for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least one week, at least two weeks, at least a month, or at least 3 months.
  • Neu5Gc competitor with at a concentration of at least 100 ⁇ , at least 200 ⁇ , at least 500 ⁇ , at least 1 mM, at least 2 mM, at least 5 mM, at least 10 mM, at least 20 mM, at least 50 mM, at least 100 mM, for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least one week, at least two weeks, at least a month, or at least 3 months.
  • the cultured cell line from which human glycosylated polypeptides in which Neu5Gc is substantially reduced include human and non-human cells including, but not limited to, hybridomas, Chinese Hamster Ovary (CHO) cells, murine myeloma cells, murine CI 27 cells, Baby Hamster Kidney (BHK) cells, HT-1080 and Human embryonic kidney cells (HEK293).
  • human and non-human cells including, but not limited to, hybridomas, Chinese Hamster Ovary (CHO) cells, murine myeloma cells, murine CI 27 cells, Baby Hamster Kidney (BHK) cells, HT-1080 and Human embryonic kidney cells (HEK293).
  • Human glycosylated polypeptides in which Neu5Gc is substantially reduced can also be produced in a non-human animal, including transgenic animals engineered to express a human gene, by supplementing their feed with a Neu5Gc competitor that can effectively compete with Neu5Gc, such as N-acetylneuraminic acid (Neu5Ac) or its precursor N-acetylmannosamine (ManNAc).
  • a Neu5Gc competitor such as N-acetylneuraminic acid (Neu5Ac) or its precursor N-acetylmannosamine (ManNAc).
  • the non-human animal is CMAH-defective. Methods for generating CMAH-defective non-human animals are disclosed in US Patent Pub. No. 2008/0166805 (USSN 11/449,167).
  • the CMAH defect is "leaky" in that residual Neu5Gc contamination of the glycopolypeptides obtained from the non-human animal is still observed. This can occur, for example, where the non-human animal has residual activity of CMAH or an alternate metabolic pathway that allows the conversion of Neu5Ac to Neu5Gc. Feeding of Neu5Gc competitor to a non-human animal can be by a large bolus or by gradual increasing concentrations.
  • Non-human animals from which can be isolated human glycosylated polypeptides in which Neu5Gc is substantially reduced include, but are not limited to, pigs, sheep, goats and cows.
  • Non-human glycosylated polypeptides in which Neu5Gc is substantially reduced can also be isolated from CMAH-defective non-human animals fed a diet that includes Neu5Gc competitor.
  • Pancreaze® for which Neu5Gc is substantially reduced, can be isolated from pig fed a diet that includes Neu5Ac or ManNAc.
  • the skilled artisan can determine the amount of Neu5Gc competitor to add to the pig feed in order to achieve substantially reduced Neu5Gc in the product isolated from the pig by the methods presented herein. Methods for correlating appropriate dosages between species are well known in the art.
  • the human glycosylated polypeptides in which Neu5Gc is substantially reduced can be isolated from tissue or from various secretions of the non-human animal, such as milk.
  • Glycosylated polypeptides of the invention can have a mol fraction of Neu5Gc of less than 2, less than 1, less than 0.5, less than 0.2, less than 0.1, less than 0.05, less than 0.02, less than 0.01, less than 0.005, less than 0.002, or less than 0.001.
  • the glycosylated polypeptides of the invention can have a mol fraction of Neu5Gc that is substantially reduced but more than 0.01, more than 0.02, more than 0.05, more than 0.1, or more than 0.5 mol fraction of Neu5Gc.
  • the absolute Neu5Gc content will also depend on the extent of glycosylation and sialylation of a given glycosylated polypeptide, which can be determined by methods disclosed herein and known in the art.
  • the Neu5Gc content of the glycosylated polypeptide can be determined in the presence and absence of Neu5Gc competitor supplement to determine how much of a reduction of mol fraction of Neu5Gc can be attained.
  • Such methods include, but are not limited to, Western blot, ELISA and immunoprecipitation using anti-Neu5Gc antibodies or HPLC determination.
  • Glycosylated polypeptides substantially reduced in Neu5Gc can include, but are not limited to, monoclonal antibodies, Fc-fusion proteins, hormones, cytokines, clotting factors, enzyme inhibitors, enzymes and antisera.
  • Tables 1 and 2, below, provide a non-exhaustive list of various commercially available recombinant therapeutic agents that when produced by the methods described herein, result in a recombinant therapeutic agent with a human-like glycosylation pattern that is substantially reduced in non-human sialic acid N-glycolylneuraminic acid (Neu5Gc). The agents are sorted generally by function in Table 1 and by cell source.
  • glycosylated polypeptides currently produced in non-animal cells can be produced by methods described herein that affords the glycosylated polypeptides human- like glycosylation patterms that are substantially reduced in Neu5Gc.
  • non-animal cells e.g. E. coli and yeast
  • Examples of recombinant human polypeptides produced in non-animal cells, and therefore lacking human-like glycosylation patterns, which could be produced in animal or human cells to obtain human-like glycosylation patterns are provided in Table 3.
  • PDGF Platelet-derived growth (yeast) factor
  • compositions of the invention can be used for the same medical indications as the corresponding Neu5Gc-contaminated glycosylated polypeptide or improperly glycosylated polypeptide (having a non-human glycosylation pattern or composition), but with a lowered immune intolerance in the human subject and/or with a potentially higher efficacy due to a lowered circulatory clearance rate.
  • Methods for detecting immune tolerance are well known in the art. For example, the interaction of glycosylated polypeptides wherein Neu5Gc has been substantially reduced can be compared to glycosylated polypeptides from non-treated sources by measuring the immunoreactivity of human serum known to have anti-Neu5Gc antibodies.
  • the relative immunoreactivity as determined above can be used to predict the relative clearance of the glycosylated polypeptide, i.e. a higher relative immunoreactivity predicts that the glycosylated polypeptide would be more rapidly cleared from the bloodstream and, therefore, more likely to have a lowered bioactivity. Consequently, glycosylated polypeptides wherein Neu5Gc has been substantially reduced will therefore likely have a longer clearance rate than the correlative Neu5Gc-contaminated glycosylated polypeptide and therefore would be predicted to have higher bioactivity.
  • the relative effect of Neu5Gc reduction on plasma clearance in humans can be assessed by, for example, (1) comparing the plasma clearance of glycosylated polypeptides wherein Neu5Gc has been substantially reduced versus glycosylated polypeptides from untreated sources or (2) comparing anti-Neu5Gc antibody levels wherein Neu5Gc has been substantially reduced versus glycosylated polypeptides from untreated sources, such as in a CM AH negative animal model, such as the CM AH null mouse.
  • compositions of the invention may be formulated as solutions or lyophilized powders for parenteral administration. Powders may be reconstituted by addition of a suitable diluent or other pharmaceutically acceptable carrier prior to use. Liquid formulations may be buffered, isotonic, aqueous solutions. Powders also may be sprayed in dry form. Examples of suitable diluents are normal isotonic saline solution, standard 5% dextrose in water, or buffered sodium or ammonium acetate solution.
  • Such formulations are especially suitable for parenteral administration, but may also be used for oral administration or contained in a metered dose inhaler or nebulizer for insufflation. It may be desirable to add excipients such as polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol, sodium chloride, sodium citrate, and the like.
  • compounds may be encapsulated, tableted or prepared in an emulsion or syrup for oral administration.
  • Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition.
  • Solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin.
  • Liquid carriers include syrup, peanut oil, olive oil, saline and water.
  • the carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax.
  • the amount of solid carrier varies but, preferably, will be between about 20 mg to about 1 g per dosage unit.
  • the pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulating, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms.
  • the preparation may be in the form of a syrup, elixir, emulsion, or an aqueous or non-aqueous suspension.
  • the invention compounds may be combined with excipients such as cocoa butter, glycerin, gelatin or polyethylene glycols and molded into a suppository.
  • Compounds may be formulated to include other medically useful drugs or biological agents.
  • the compounds also may be administered in conjunction with the administration of other drugs or biological agents useful for the disease or condition to which the invention compounds are directed.
  • an effective amount refers to a dose sufficient to provide concentrations high enough to impart a beneficial effect on the recipient thereof.
  • the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated, the severity of the disorder, the activity of the specific compound, the route of administration, the rate of clearance of the compound, the duration of treatment, the drugs used in combination or coincident with the compound, the age, body weight, sex, diet, and general health of the subject, and like factors well known in the medical arts and sciences.
  • a compound can be administered parenterally, such as intravascularly, intravenously, intraarterially, intramuscularly, subcutaneously, or the like. Administration can also be orally, nasally, rectally, transdermally or inhalationally via an aerosol.
  • the compound may be administered as a bolus, or slowly infused.
  • a therapeutically effective dose can be estimated initially from cell culture assays by determining an IC50.
  • a dose can then be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 as determined in cell culture. Such information can be used to more accurately determine useful initial doses in humans.
  • Levels of drug in plasma may be measured, for example, by HPLC. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.
  • mice Cmah null mice 6 , and were backcrossed to C57B1/6 mice for >10 generations.
  • periodate and borohydride were pre-mixed and then added to the wells (the borohydride inactivates the periodate).
  • the borohydride inactivates the periodate.
  • wells were then washed 3 times with 100 mM sodium acetate, 100 mM NaCl, pH 5.5 before further analysis.
  • Binding of IgY was detected using HRP-conjugated donkey anti- chicken IgY antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) (1 :50,000 in TBST) and development with O-phenylenediamine in citrate- phosphate buffer, pH 5.5, with absorbance being measured at 495 nm.
  • ELISA samples were studied at least in triplicate. Similar to the ELISA with the anti-Neu5Gc chicken IgY, human anti-Neu5Gc IgG that had been purified from the serum of healthy humans 7 and biotinylated was also used as the primary antibody (1 : 100 in TBST).
  • Binding of the human antibodies to the therapeutic antibodies was detected using HRP-conjugated Streptavidin (1 : 10,000) (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) followed by development as described above. Samples were studied in triplicate.
  • Binding of the chicken anti-Neu5Gc IgY was detected using an HRP-conjugated donkey anti-chicken IgY antibody for 1 h (1 :50,000 in TBST), followed by incubation with SuperSignal West Pico Substrate (Thermo Fisher Scientific, Waltham, MA) as per manufacturer's recommendation, exposed to X-ray film and the film developed. Similar to the Western blot with the chicken anti- Neu5Gc IgY, purified biotinylated human anti-Neu5Gc IgG was also used as the primary antibody (1 : 100 in TBST). Binding of the human antibodies to the therapeutic antibodies was detected using HRP-conjugated Streptavidin (1 : 10,000 in TBST) followed by development as described above.
  • CIC-Clq Binding Assay Immune complex formation was detected by using the CIC (C1Q) ELISA Kit (BTJHLMA N Laboratories AG, Schonenbuch, Switzerland) as described in the manufacturers 's guidelines . Briefly, 100 ⁇ of human serum with low or high anti-Neu5Gc antibodies (S30 and S34, respectively, from Ref. 7) was incubated with 40 ⁇ g of Cetuximab or Panitumumab for 14 h at 4°C. 1 :50 dilutions of the mix were applied to human Clq coated ELISA wells, and incubated for 1 h at 25 °C. Binding was detected using alkaline phosphatase conjugated Protein A. After another washing step, the enzyme substrate (para-nitrophenyl- phosphate) was added followed by a stopping step. The absorbance was measured at 405 nm. Samples were studied in triplicate.
  • DMB-HPLC DMB-HPLC. Samples were subjected to base treatment with 0.1 M NaOH (final) at 37°C for 30 min to remove O-acetyl esters, and the sialic acids were released by acid hydrolysis with 2 M acetic acid (final), at 80°C for 3 hours. Samples were passed through Microcon-10 columns (20 min, 10000 x g; Millipore), derivatized with l,2-diamino-4,5-methyl dioxybenzene (DMB), and analyzed by HPLC as described 9 in Hara et al., 1986, J. Chromatography 377: 111- 119. Measured values of non acid-treated controls (free sialic acids) were subtracted.
  • Binding of mouse IgG was detected by using HRP-conjugated goat anti-mouse IgG-Fc (Jackson ImmunoResearch Laboratories, Inc.; West Grove, PA) (1 : 10,000 in PBST) and development with O-phenylenediamine in citrate-phosphate buffer, pH 5.5, with absorbance being measured at 490 nm.
  • ELISA samples were studied in triplicate.
  • wells were coated with human or bovine fibrinogen, which carry Neu5Ac or Neu5Gc on otherwise identical N- glycans 11 .
  • Wells were then blocked with TBST for 2 h followed by incubation with 1 : 100 dilutions of the mouse sera. Binding of the mouse antibodies was detected by using HRP- conjugated goat anti-mouse IgG Fc fragment antibody (1 : 10000 in TBST).
  • the cells were harvested at day 1, 2, 3, 4 and 5 by scraping into the culture media, collecting by pelleting, washing once with PBS, transferring to a 1.5 ml microcentrifuge tube, pelleting and freezing the cell pellet.
  • All collected cell pellets were homogenized in 300 ⁇ of ice-cold 20 mM potassium phosphate pH 7 using 3-20 sec burst with a Fisher Sonicator.
  • Glycoconjugate -bound Sias were precipitated by adding 700 ⁇ 1 of 100% ice-cold ethanol (final 70% ethanol) and incubating at -20°C overnight.
  • the samples were spun at 20000 X g for 15 min and the supernatants transferred to clean tubes and dried on a speed vac.
  • the precipitated glycoconjugates and dried ethanol supernatants were each suspended in 100 ⁇ of 20 mM potassium phosphate pH 7 by sonication.
  • Sias were released from both fractions by acid hydrolysis with 2 M acetic acid (final) and incubating at 80°C for 3 h.
  • Samples were passed through a Microcon-10 filter and the filtrate derivatized with DMB reagent, for analysis of Sias by HPLC.
  • Sialic Acid Specificity of Anti-Neu5Gc IgY Binding Sialic acid-specificity of anti- Neu5Gc IgY binding was reaffirmed by pre-treatment with mild sodium periodate, under conditions that selectively cleave Sia side chains (Figure 1C), and abolish reactivity of such antibodies 15 ' 5 .
  • Cet and Pan were used for coating, then blocked, and sialic acid epitopes eliminated chemically using sodium metaperiodate. The reaction was stopped with sodium borohydride. As a control, periodate and borohydride were pre-mixed and then added to the wells (the borohydride inactivates the periodate).
  • ELISA samples were studied at least in triplicate and data shown are Mean +/- SD. ***p ⁇ 0.001, Paired Two-tailed t-test.
  • Sialic Acid Quantity The levels of Sialic acids on the therapeutic antibodies (TAbs) Cet and Pan were determined. Cet and Pan were treated with sialidase or heat-inactivated sialidase as for Figure 1A and used for coating ELISA wells, then blocked and incubated with human anti-Neu5Gc IgG that had been purified from the serum of healthy humans and biotinylated (Figure ID). Samples were studied in triplicate and data shown as Mean +/- SD. ***p ⁇ 0.001 Panitumumab carries 0.22 mol/mol of Sias, with ⁇ 0.1% Neu5Gc. In contrast, Cetuximab carries 1.84 mol/mol of Sias, mostly as Neu5Gc.
  • Cetuximab formed immune complexes in a human serum with high levels of anti-Neu5Gc antibodies (serum S34, from reference 7), and not with the low titer serum (serum S30, from reference 7). In contrast, Panitumumab gave no detectable immune complex formation with either sera.
  • ECF extracellular lung disease .
  • Sera pooled from naive, control-immunized or Neu5Gc-immunized syngeneic mice were passively transferred via intraperitoneal injection, ensuring equal starting concentrations of circulating anti-Neu5Gc antibodies.
  • Anti-Neu5Gc IgG levels in the pooled sera from Neu5Gc-immunized mice were quantified by ELISA with a Neu5Gca2-6Gaipi-4Glc-conjugate as a target, as previously described 7 (97.5 ⁇ g/ml).
  • the amount of pooled antibody injected was calculated to achieve an approximate starting concentration of 4 ⁇ g/ml IgG in the ECF of these mice, i.e. ⁇ 4 times excess of anti-Neu5Gc antibodies compared to the drug in mice, and similar to levels found in some humans 7 .
  • Mice were bled periodically after the passive transfer of mouse serum.
  • mice To exclude any impact of the partly (Cetuximab) or fully human protein portion (Panitumumab) in mice, murine IgG was also injected as a positive control, as it happens to carry primarily Neu5Gc. Cetuximab and murine IgG (but not Panitumumab) induced a Neu5Gc- specific IgG immune response (Figure 2B).
  • the percent Neu5Gc shown is the amount of Neu5Gc relative to the total sialic acids.
  • Neu5Ac addition resulted in more rapid disappearance of ethanol-precipitable (glycosidically-bound) Neu5Gc from the cells and also from secreted glycoproteins ( Figure 3A-B).
  • Neu5Ac addition to the medium eliminates or reduces Neu5Gc contamination of human cells.
  • the individually collected media was centrifuged to remove cell debris and adjusted to 5 mM Tris-HCl pH 8.
  • the fusion protein was purified using Protein-A Sepharose. Sialic acid content was determined by DMB-HPLC analysis (Figure 3C). The area under each peak was obtained and the percent of Neu5Gc in each sample was determined relative to Neu5Ac.
  • Total cell membranes from the same CHO cells were prepared and used for DMB-HPLC analysis ( Figure 3D). CHO membrane proteins from the above experiments were separated by SDS-PAGE and transferred onto nitrocellulose membranes ( Figure 3E). The expression of Neu5Gc was detected by incubating with polyclonal affinity purified chicken anti-Neu5Gc antibody.
  • Hybridoma cell lines that express monoclonal antibodies specific to human epidermal growth factor receptor are disclosed in U.S. Patent No. 6,217,866, incorporated by reference herein, the preparation of which is modified as follows:
  • the 108 IgG2a hybridoma cell line is generated by immunizing mice with CH 71 cells expressing the EGF receptor and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 5 mM Neu5Ac depleted of complement activity by incubation at 56°C. for 30 minutes and grown in glutamine, penicillin, streptomycin and sodium pyruvate, at 37°C. in 5% C02 : 95% air atmosphere.
  • the 96 IgM hybridoma cell line is generated by the same procedure as that described for the 108 IgG2a hybridoma cell line.
  • the medium wherein the monoclonal antibodies are secreted is separated from the hybridoma cells.
  • Monoclonal antibodies are precipitated by slow addition of saturated ammonium sulfate at 4°C to a final concentration of 45% (v/v), pH 7.5, for 24 hours.
  • the precipitate is collected by centrifugation at 10,000 g for 15 minutes and washed twice with 50% v/v ammonium sulfate, pH 7.5. at 4°C. Further purification is carried out by affinity chromatography on Sepharose CL protein A (Pharmacia) in 0.14M Tris buffer, pH 8.0 and the 108 monoclonal antibody is eluted with 0.1M citrate buffer, pH 3.0, followed by extensive dialysis against PBS.
  • the F(ab)'2 (2 mg/ml) is reduced by 10 mM dithiothreitol in 20 mM Tris buffer, pH 8.2, for 1 hour at 37°C. Alkylation is performed in 40 mM iodoacetamide for 30 minutes at 37°C, followed by extensive dialysis against PBS at 4°C. Purity and complete digestion of the various fragments
  • the protein dimer of hFSH contains 2 polypeptide units, labeled alpha and beta subunits.
  • the alpha subunits of leutenizing hormone (LH), FSH, thyroid stimulating hormone (TSH), and human chorionic gonadotrophin (hCG) are identical, and contain 92 amino acids.
  • the beta subunits vary.
  • FSH has a beta subunit of 118 amino acids (FSHB), which confers its specific biologic action and is responsible for interaction with the FSH-receptor.
  • expression plasmid pRF375 which expresses the alpha subunit under mouse metallothienine control
  • expression plasmid CL28FSH2.8BPV which expresses the beta subunit, as well as their coexpression in mouse C127 cells
  • U.S. Patent No. 5,767,251 is herein incorporated by reference.
  • Glycosylation mutants of hFSH as well as coexpression of alpha and beta subunits in CHO cells are disclosed in U.S. Patent No. 7,700,112, incorporated by reference herein.
  • hFSH is produced by growing stably transfected cells in two 850 cm roller bottles.
  • Production media containing hFSH is harvested and filtered using 0.22 ⁇ filter units and frozen at -70°C.
  • the target proteins in media are thawed overnight at 4°C, and concentrated by ultrafiltration using Ultrasette Screen Channel TFF device, 10 K Omega membrane, P/N 0S010C70 (Pall Life Science).
  • the retentate is recovered and dialyzed overnight versus 0.1 M Tris, pH 7.4 containing 0.5 M NaCl, 3 x 5 Liters.
  • the dialyzed protein is recovered, 0.22 ⁇ filtered, and purified immediately or stored at -70°C until purified.
  • Immunoaffinity Purification (Alternate Purification). Human follicle stimulating hormone is purified by anti FSH immunoaffinity resin B5 (Serobio) containing 2.2 mg anti-FSH antibody per ml of resin. A 10.2 ml bed volume is prepared in 1.5 cm x 10 cm OmniFit column. The resin is pre equilibrated with 0.1 M Tris, pH 7.4 containing 0.5 M NaCl. The dialyzed crude protein is loaded at one ml/min.
  • the column is washed sequentially with the five column volumes of 0.1 M Tris, pH 7.4 containing 0.5 M NaCl, five column volumes of 100 mM ammonium bicarbonate, pH 7.6, and the target protein eluted with 18-20 column volumes of 1 M NH40H.
  • the fractions containing the eluted protein are pooled, neutralized with glacial acetic acid, and concentrated by ultrafiltration with Amicon stirred cell using Amicon YM 10 membrane.
  • the retentate is dialyzed in Pierce Snakeskin dialysis tubing, 10 K MWCO, versus 4 x 5 liters of water over 24 hours.
  • the dialyzed protein is recovered and concentrated by Centriprep YM 10 to decrease the volume to approximately one ml.
  • r-hPC Recombinant human protein C
  • r-hPC Recombinant human protein C
  • AVI 2 Recombinant human protein C
  • Human Embryonic Kidney Cell Line 293 is available from the American Type Culture Collection under the accession number ATCC CRL 1573 and the adenovirus-trans formed Syrian hamster cell line AVI 2 is available from the American Type Culture Collection under the accession number ATCC CRL 9595.
  • the transformation procedure is described, for example, in U.S. Pat. No. 4,992,373 where the plasmid encoding r-hAPC is transfected into 293 cells, then stable transformants are identified, subcultured and grown in serum-free media supplemented with 5 mM Neu5Ac. After fermentation, cell-free medium was obtained by microfiltration.
  • r-hPC Recombinant human protein C
  • Human protein C is separated from the culture fluid by an adaptation of the techniques of Yan, U.S. Pat. No. 4,981,952.
  • the clarified medium is made 4 mM in EDTA before it is absorbed to an anion exchange resin (Fast-Flow Q, Pharmacia).
  • an anion exchange resin Frazier-Flow Q, Pharmacia.
  • the bound recombinant human protein C zymogen is eluted with 20 mM Tris, 150 mM NaCl, 10 mM CaCl 2 , pH 7.4. Purity is judged by SDS-polyacrylamide gel electrophoresis.
  • the eluted protein is prepared for activation by removal of residual calcium by passing over a metal affinity column (Chelex-100, Bio-Rad) to remove calcium and again bound to an anion exchanger (Fast Flow Q, Pharmacia). Both of these columns are arranged in series and equilibrated in 20 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 7.4. Following loading of the protein, the Chelex-100 column is washed with one column volume of the same buffer before disconnecting it from the series.
  • r-hPC Recombinant human protein C
  • r-hPC Recombinant human protein C
  • Bovine thrombin as described in Carlson, et al., U.S. Pat. No. 6,159,468.
  • Bovine thrombin is coupled to Activated CH-Sepharose 4B (Pharmacia) in the presence of 50 mM HEPES, pH 7.5 at 40°C.
  • the coupling reaction is done on resin already packed into a column using approximately 5000 units thrombin/mL resin.
  • the thrombin solution is circulated through the column for approximately 3 hours before adding 2-aminoethanol (MEA) to a concentration of 0.6 mL/L of circulating solution.
  • MEA 2-aminoethanol
  • the MEA-containing solution is circulated for an additional 10-12 hours to assure complete blockage of the unreacted amines on the resin.
  • the thrombin-coupled resin is washed with 10 column volumes of 1 M NaCl, 20 mM Tris, pH 6.5 to remove all non-specifically bound protein, and is used in activation reactions after equilibrating in activation buffer.
  • Purified r-hPC is made 5 mM in EDTA (to chelate any residual calcium) and diluted to a concentration of 2 mg/mL with 20 mM Tris, pH 7.4 or 20 mM Tris-acetate, pH 6.5. This material is passed through a thrombin column equilibrated at 37°C with 50 mM NaCl and either 20 mM Tris pH 7.4 or 20 mM Tris-acetate pH 6.5. The flow rate is adjusted to allow for approximately 20 min. of contact time between the r-hPC and thrombin resin. The effluent is collected and immediately assayed for amidolytic activity.
  • the material does not have a specific activity (amidolytic) comparable to an established standard of aPC, it is recycled over the thrombin column to activate the r-hPC to completion. This is followed by 1 : 1 dilution of the material with 20 mM buffer as above, with a pH of either 7.4 or 6.5 to keep the aPC at lower concentrations while it awaits the next processing step.
  • Removal of leached thrombin from the aPC material is accomplished by binding the aPC to an anion exchange resin (Fast Flow Q, Pharmacia) equilibrated in activation buffer (either 20 mM Tris, pH 7.4 or 20 mM Tris-acetate, pH 6.5) with 150 mM NaCl. Thrombin does not interact with the anion exchange resin under these conditions, but passes through the column into the sample application effluent.
  • activation buffer either 20 mM Tris, pH 7.4 or 20 mM Tris-acetate, pH 6.5
  • a 2-6 column volume wash with 20 mM equilibration buffer is done before eluting the bound aPC with a step elution using 0.4 M NaCl in either 5 mM Tris-acetate, pH 6.5 or 20 mM Tris, pH 7.4. Higher volume washes of the column facilitate more complete removal of the dodecapeptide.
  • the material eluted from this column is stored either in a frozen solution (-20°C) or as a lyophilized powder.
  • the anticoagulant activity of activated protein C is determined by measuring the prolongation of the clotting time in the activated partial thromboplastin time (APTT) clotting assay.
  • a standard curve is prepared in dilution buffer (1 mg/mL radioimmunoassay grade bovine serum albumin [BSA], 20 mM Tris, pH 7.4, 150 mM NaCl, 0.02% NaN.sub.3) ranging in protein C concentration from 125-1000 ng/niL, while samples are prepared at several dilutions in this concentration range.
  • a fermenter is seeded with a factor VIII expressing clone with the cells at a density of about 3x106 cells/ml.
  • the fermenter is perfused at a rate of 4 volumes per day with the serum- free production medium as described in the preceding paragraph.
  • a final cell density of 2xl0 7 cells/ml is sustained throughout the evaluation period (45 days).
  • factor VIII expressing clone is perfused with the serum free production medium supplemented with Plasmanate® (Human plasma albumin, Talecris, Research Triangle Park, NC) HPP fraction and 5 mM Neu5Ac, and is able to sustain high productivity.
  • Plasmanate® Human plasma albumin, Talecris, Research Triangle Park, NC
  • the cells are perfused with the same serum free production medium containing and 5 mM Neu5Ac but without Plasmanate® HPP fraction.
  • the cells continue to produce high levels of FVIII in a plasma derived protein-free environment.
  • Plasma derived protein-free means that essentially no proteins isolated from plasma is added to the medium.
  • CHOEFI2S-9 cells are inoculated into two 2-layer cell factories (NUNC, 1200 cm 2 ) in Ham's F12, 10% v/v FCS, and 5 mM Neu5Ac (N-acetylneuraminic acid) and antibiotics. Cells are grown to confluency, the medium removed and the cells are then rinsed 3 -times with PBS and re-fed with 200 ml of Ham's F12 without FCS but supplemented with 5 mM Neu5Ac, antibiotics and 10 mM-NH4Cl.
  • NUNC 2-layer cell factories
  • the medium After 4 days in culture, the medium are collected and replaced with Ham's F12, 10% v/v FCS, PSK, and 5 mM Neu5Ac but without NH 4 C1 for 3 days. This cycle is repeated several times.
  • the conditioned serum free Ham's F12 medium supplemented with NH 4 C1 and 5 mM Neu5Ac is collected, clarified by filtration (0.2 ⁇ filture; Millipore) and stored at 4°C.
  • the recombinant Idursulfase is purified from the collected medium by a 3-step column procedure.
  • the medium is dialysed overnight at 4°C against 30 mM-Tris/HCl, pH 7.0/10% v/v glycerol/0.1 mM-DTE/3 mM-NaN 3 (buffer A) and is applied to a PBE94 column (8 cm x 1.5 cm) equilibrated in buffer A (flow-rate 1.0 ml/min) and then washed with 100 ml of buffer A.
  • Bound proteins are diluted with polybuffer 74 that has been diluted 1 : 18 with water, the H adjusted to 4.0 with HC1 and the solution made 10% v/v in glycerol, 0.1 mM-DTE and 3 mM-NaN 3 .
  • the column is further eluted with 100 ml 15 mM-ditheriothreitol/3 mM-NaN 3 (buffer B).
  • the rIDS eluted in buffer B is applied at a flow-rate of 1.0 ml/min to a Blue A agarose column (6 cm x 0.7 cm) also equilibrated in buffer B.
  • the rIDS activity from this step is applied in 1.0 ml volumes to an LKB Ultrachrom GTi f.p.l.c. system with a TSK G3000SW Ultrapac column (30 cm x 0.8 cm) equilibrated and eluted in buffer B at a flow-rate of 0.5 ml/min and pressure of 150 kPa. Fractions containing rIDS activity are pooled and analysed under denaturing and nondenaturing condition on SDS-PAGE (10% w/v acrylamide) to estimate apparent subunit size.
  • Varki A. Glycan-based interactions involving vertebrate sialic-acid-recognizing proteins. Nature 446, 1023-1029 (2007).

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Abstract

La présente invention concerne des compositions polypeptidiques glycosylées ayant une teneur en Neu5Gc sensiblement réduite. Les compositions polypeptidiques glycosylées ayant une teneur en Neu5Gc sensiblement réduite peuvent être obtenues à partir de sources cellulaires mises en culture avec un compétiteur de Neu5Gc ou à partir de sources animales non humaines nourries selon un régime supplémenté en compétiteur de Neu5Gc. La présente invention concerne également des méthodes de traitement d'un sujet humain par lesdites compositions.
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WO2017044811A1 (fr) * 2015-09-11 2017-03-16 Bruce Andrien Variants d'éculizumab et d'éculizumab glycosylé de recombinaison
WO2018024770A1 (fr) 2016-08-03 2018-02-08 Formycon Ag Production d'ustekinumab biosimilaire dans des cellules cho
CN114167060A (zh) * 2021-11-25 2022-03-11 辽宁师范大学 一种肝癌生物标志物及检测肝癌的试剂盒

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* Cited by examiner, † Cited by third party
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US20090099073A1 (en) * 2001-12-21 2009-04-16 Human Genome Sciences, Inc. Albumin Fusion Proteins
WO2010030666A2 (fr) * 2008-09-09 2010-03-18 The Regents Of The University Of California Élimination d’un acide sialique non-humain contaminant par compétition métabolique

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090099073A1 (en) * 2001-12-21 2009-04-16 Human Genome Sciences, Inc. Albumin Fusion Proteins
WO2010030666A2 (fr) * 2008-09-09 2010-03-18 The Regents Of The University Of California Élimination d’un acide sialique non-humain contaminant par compétition métabolique

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017044811A1 (fr) * 2015-09-11 2017-03-16 Bruce Andrien Variants d'éculizumab et d'éculizumab glycosylé de recombinaison
JP2018528218A (ja) * 2015-09-11 2018-09-27 アンドリアン、ブルース 組換えグリコシル化エクリズマブおよびエクリズマブ変異体
JP2021183611A (ja) * 2015-09-11 2021-12-02 アレクシオン ファーマシューティカルズ， インコーポレイテッド 組換えグリコシル化エクリズマブおよびエクリズマブ変異体
WO2018024770A1 (fr) 2016-08-03 2018-02-08 Formycon Ag Production d'ustekinumab biosimilaire dans des cellules cho
US20220204607A1 (en) * 2016-08-03 2022-06-30 Fyb 202 Project Gmbh Production of Biosimilar Ustekinumab In CHO Cells
US12098196B2 (en) * 2016-08-03 2024-09-24 Fyb202 Project Gmbh Production of biosimilar ustekinumab in CHO cells
CN114167060A (zh) * 2021-11-25 2022-03-11 辽宁师范大学 一种肝癌生物标志物及检测肝癌的试剂盒

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