WO2022040073A1 - Nouvelles méthodes de traitement de neutropénie au moyen d'un complexe protéique à g-csf - Google Patents

Nouvelles méthodes de traitement de neutropénie au moyen d'un complexe protéique à g-csf Download PDF

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WO2022040073A1
WO2022040073A1 PCT/US2021/046108 US2021046108W WO2022040073A1 WO 2022040073 A1 WO2022040073 A1 WO 2022040073A1 US 2021046108 W US2021046108 W US 2021046108W WO 2022040073 A1 WO2022040073 A1 WO 2022040073A1
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Prior art keywords
immunoglobulin
region
csf
protein complex
peptidyl polymer
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PCT/US2021/046108
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English (en)
Inventor
Gajanan Bhat
Shanta Chawla
Jae Hyuk CHOI
Eun Jung KIM
Yu Yon KIM
Gyu Hyan LEE
Hyesun Han
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Spectrum Pharmaceuticals, Inc.
Hanmi Pharm Co., Ltd.
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Priority claimed from US16/996,635 external-priority patent/US11684655B2/en
Application filed by Spectrum Pharmaceuticals, Inc., Hanmi Pharm Co., Ltd. filed Critical Spectrum Pharmaceuticals, Inc.
Priority to CA3189970A priority Critical patent/CA3189970A1/fr
Publication of WO2022040073A1 publication Critical patent/WO2022040073A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
    • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention relates to protein complexes, pharmaceutical compositions, and methods of use thereof for treating, preventing, or reducing the risk of developing a condition, such as neutropenia.
  • the protein complex can be formed by linking an immunoglobulin Fc region to a physiologically active polypeptide via a non-peptidyl polymer, in which the non-peptidyl polymer is linked to the immunoglobulin Fc region.
  • Neutropenia is a relatively common disorder most often due to chemotherapy treatments, adverse drug reactions, or autoimmune disorders.
  • Chemotherapy-induced neutropenia is common toxicity caused by the administration of anticancer drugs. It is associated with life-threatening infections and may alter the chemotherapy schedule, thus impacting early and long-term outcomes.
  • Febrile Neutropenia (FN) is major dose-limiting toxicity of myelosuppressive chemotherapy regimens such as docetaxel, doxorubicin, cyclophosphamide (TAC); dose-dense doxorubicin plus cyclophosphamide (AC), with or without subsequent weekly or semiweekly paclitaxel; and docetaxel plus cyclophosphamide (TC). It usually leads to prolonged hospitalization, intravenous administration of broad-spectrum antibiotics and is often associated with significant morbidity and mortality. About 25% to 40% of treatment naive patients develop febrile neutropenia with common chemotherapy regimens.
  • G-CSF granulocyte colony-stimulating factor
  • antibiotic agents to combat this condition.
  • G-CSF or its other polypeptide derivatives are easy to denature or easily de-composed by proteolytic enzymes in blood to be readily removed through the kidney or liver. Therefore, to maintain the blood concentration and titer of the G-CSF- containing drugs, it is necessary to frequently administer the protein drug to patients, which causes excessive suffering in patients.
  • G-CSF was chemically attached to polymers having a high solubility, such as polyethylene glycol (“PEG”), thereby increasing its blood stability and maintaining suitable blood concentration for a longer time.
  • PEG polyethylene glycol
  • Filgrastim, tbo-filgrastim, and Pegfilgrastim are G-CSFs currently approved by the US Food and Drug Administration (FDA) for the prevention of chemotherapy-induced neutropenia; while the European guidelines also include lenograstim as a recommended G-CSF in solid tumors and non-myeloid malignancies, it is not approved for use in the US. Binding of PEG to G-CSF, even though it may increase blood stability, does dramatically reduce the titer needed for optimal physiologic effect.
  • this disclosure addresses the need mentioned above in a number of aspects.
  • this disclosure provides a method of preventing, alleviating or treating a condition in a subject in need thereof, wherein the condition is characterized by compromised white blood cell production in the subject.
  • the method comprises administering to the subject a therapeutically effective amount of a chemotherapeutic regimen followed by a therapeutically effective amount of a protein complex comprising a modified human granulocyte-colony stimulating factor (hG-CSF) covalently linked to an immunoglobulin Fc region via a non-peptidyl polymer, wherein the non- peptidyl polymer is site-specifically linked to an N-terminus of the immunoglobulin Fc region, wherein the modified hG-CSF comprises substitutions at positions Cysl7 and Pro65, and wherein the protein complex is administered on the same day as the chemotherapeutic regimen.
  • hG-CSF human granulocyte-colony stimulating factor
  • this disclosure provides a method for increasing stem cell production in a donor.
  • the method comprises administering to the subject a therapeutically effective amount of a chemotherapeutic regimen followed by a therapeutically effective amount of a protein complex comprising a modified hG-CSF covalently linked to an immunoglobulin Fc region via a non- peptidyl polymer, wherein the non-peptidyl polymer is site-specifically linked to an N-terminus of the immunoglobulin Fc region, wherein the modified hG-CSF comprises substitutions at positions Cysl7 and Pro65, and wherein the protein complex is administered on the same day as the chemotherapeutic regimen.
  • the condition is selected from reduced hematopoietic function, reduced immune function, reduced neutrophil count, reduced neutrophil mobilization, mobilization of peripheral blood progenitor cells, sepsis, severe chronic neutropenia, bone marrow transplants, infectious diseases, leucopenia, thrombocytopenia, anemia, enhancing engraftment of bone marrow during transplantation, enhancing bone marrow recovery in treatment of radiation, chemical or chemotherapeutic induced bone marrow aplasia or myelosuppression, and acquired immune deficiency syndrome.
  • myelosuppression is neutropenia.
  • neutropenia is febrile neutropenia.
  • the compromised white blood cell production is a result of chemotherapy, radiation therapy, or idiopathic thrombocytopenic purpura.
  • the protein complex is administered after the subject is treated with adjuvant or neoadjuvant chemotherapy. In some embodiments, the protein complex is administered between 1 and 5 days after the subject is treated with adjuvant or neoadjuvant chemotherapy.
  • the adjuvant or neoadjuvant chemotherapy is a combination of docetaxel and cyclophosphamide.
  • a second dose of the protein complex is administered between 15 and 25 days after a first dose of the protein complex is administered to the subject.
  • the therapeutically effective amount of the protein complex is a unit dosage between about 5 ug/kg and about 200 pg/kg. In some embodiments, the therapeutically effective amount of the protein complex is a unit dosage form selected from: about 9 pg/kg, about 25 pg/kg, about 26 pg/kg, about 50 pg/kg, about 52 pg/kg, about 88 pg/kg, about 100 pg/kg, about 150 pg/kg, and about 200 pg/kg.
  • the therapeutically effective amount of the protein complex is 13.2 mg of the protein complex in a 0.6 mb dosage volume.
  • the method further comprises administering to the subject a therapeutically effective amount of a second agent.
  • the second agent comprises an anti-cancer agent.
  • the substitution at Cysl7 is Cysl7Ser. In some embodiments, the substitution at Pro65 is Pro65Ser. In some embodiments, the modified human G-CSF comprises the amino acid sequence of SEQ ID NOs: 1. In some embodiments, the immunoglobulin Fc region comprises the amino acid sequence of SEQ ID NO: 2.
  • both ends of the non-peptidyl polymer are respectively linked to the modified human G-CSF and the immunoglobulin Fc region through reactive groups by a covalent bond.
  • the immunoglobulin Fc region is aglycosylated. In some embodiments, the immunoglobulin Fc region consists of one to four domains selected from CHI, CH2, CH3, and CH4 domains. In some embodiments, the immunoglobulin Fc region further comprises a hinge region. In some embodiments, the immunoglobulin Fc region is an immunoglobulin Fc fragment derived from IgG, IgA, IgD, IgE, or IgM.
  • each domain of the immunoglobulin Fc fragment is a hybrid of domains, in which each domain has a different origin derived from immunoglobulins selected from IgG, IgA, IgD, IgE, and IgM.
  • the immunoglobulin Fc fragment is a dimer or multimer consisting of single chain immunoglobulins comprising domains having the same origin.
  • the immunoglobulin Fc fragment is an IgG4 Fc fragment.
  • the immunoglobulin Fc fragment is a human aglycosylated IgG4 Fc fragment.
  • the non-peptidyl polymer is selected from polyethylene glycol, polypropylene glycol, an ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, a biodegradable polymer, a lipid polymer, chitin, hyaluronic acid, and a combination thereof.
  • the non-peptidyl polymer is polyethylene glycol.
  • the polyethylene glycol has a molecular weight of about 3.4 kDa.
  • the reactive group of the non-peptidyl polymer is selected from an aldehyde group, a maleimide group, and a succinimide derivative.
  • the non- peptidyl polymer has an aldehyde group as a reactive group at both ends.
  • the non-peptidyl polymer has an aldehyde group and a maleimide group as a reactive group at both ends, respectively.
  • the non-peptidyl polymer has an aldehyde group and a succinimide group as a reactive group at both ends, respectively.
  • this disclosure provides a method for treating or preventing neutropenia in a patient diagnosed with breast cancer.
  • the method comprises administering a chemotherapy regimen of docetaxol and cyclophosphamide and a protein complex comprising a physiologically active polypeptide linked to an immunoglobulin Fc region via a non-peptidyl polymer, wherein the non-peptidyl polymer is site-specifically linked to an N-terminus of the immunoglobulin Fc region, and wherein both ends of the non-peptidyl polymer are respectively linked to the physiologically active polypeptide and the immunoglobulin Fc region through reactive groups by a covalent bond.
  • the physiologically active polypeptide is G-CSF. In some embodiments, the physiologically active polypeptide is a modified human G-CSF comprising substitutions at positions Cysl7 and Pro65. In some embodiments, wherein the modified human G-CSF comprises the amino acid sequence of SEQ ID NO: 1.
  • the immunoglobulin Fc region consists of one to four domains selected from CHI, CH2, CH3, and CH4 domains. In some embodiments, the immunoglobulin Fc region further comprises a hinge region. In some embodiments, each domain of the immunoglobulin Fc fragment is a hybrid of domains, in which each domain has a different origin derived from immunoglobulins selected from IgG, IgA, IgD, IgE, and IgM.
  • the immunoglobulin Fc region comprises an IgG4 Fc fragment; optionally wherein the immunoglobulin Fc region is aglycosylated. In some embodiments, the immunoglobulin Fc region comprises the amino acid sequence of SEQ ID NO: 2.
  • the non-peptidyl polymer is selected from polyethylene glycol, polypropylene glycol, an ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, a biodegradable polymer, a lipid polymer, chitin, hyaluronic acid, and a combination thereof.
  • the non-peptidyl polymer is polyethylene glycol.
  • the polyethylene glycol has a molecular weight of about 3.4 kDa.
  • At least one dose of the protein complex is administered to the patient within about 24 hours, about 6 hours, about 5 hours, about 3 hours, about 2 hours, about 1 hour, about 30 minutes, about 15 minutes, or about 5 minutes of the completion of chemotherapy.
  • FIG. 1A shows the results of SDS-PAGE and western blotting of a 17,65 Ser-G-CSF-PEG- Fc complex, which was prepared by N-terminal reaction of an immunoglobulin Fc region.
  • FIG. IB shows a result of peptide mapping for analyzing Fc region N-terminal binding of a 17,65 Ser-G-CSF-PEG-Fc complex, which was prepared by N-terminal reaction of an immunoglobulin Fc region.
  • FIG. 2 shows that a lower incidence of severe neutropenia in the 17,65 Ser-G-CSF-PEG-Fc (Eflapegrastim ) arm is statistically significant.
  • FIG. 3 shows that neutropenic complications, including hospitalizations due to severe neutropenia and/or use of anti-infective for neutropenia, are significantly less in the Eflapegrastim arm.
  • FIGS. 4A, 4B, 4C, 4D, and 4E are a set of graphs showing binding of Eflapegrastim to Fey receptors and Clq. Binding of Eflapegrastim to purified Fey receptors Fey RI (FIG. 4A), Fey RIIB (FIG. 4B), and Fey RIIIA (FIG. 4C), and Fey receptors on U937 cells
  • FIG. 4 (FIG. 4D) and Clq (FIG. 4E) was studied by ELISA. The concentration of individual test articles was calculated based on the theoretical Fc protein to make equivalent molarity. Mean OD values from duplicate samples are presented. The error bars represent SEM values.
  • OD Optical density
  • IVIG intravenous immunoglobulin G (immunoglobulin G)
  • GlycoFcGl glycosylated Fc fragment of human IgGl
  • AglycoFcGl Aglycosylated Fc fragment of human IgGl.
  • FIGS. 5A and 5B are a set of graphs showing FcRn binding and FcRn mediated Transcytosis. Binding of Eflapegrastim to FcRn was studied by ELISA (FIG. 5 A). The concentration of individual test articles was calculated based on the theoretical Fc protein to make equivalent molarity. For studying transcytosis (FIG. 5B), the quantity of Eflapegrastim and Pegfilgrastim transported across the cell layer was determined by ELISA. The data are presented as mean values from duplicate samples. The error bars represent SEM values. *p ⁇ 0.05; **p ⁇ 0.01. OD: optical density.
  • FIGS. 6A, 6B, and 6C are a set of graphs showing efficacy in neutropenic rats following administration of Eflapegrastim and Pegfilgrastim 24 hours after CPA chemotherapy.
  • Rats were administered with cyclophosphamide (50 mg/kg) to induce neutropenia and treated with Eflapegrastim or Pegfilgrastim 24 hours later.
  • Blood samples were collected from jugular vein, and absolute neutrophil count (ANC) was determined using hematology analyzer.
  • FIG. 6A shows the ANC profile.
  • FIG. 6B shows area under the ANC versus. Time curve above baseline (AUECANC).
  • FIG. 6C shows the duration of neutropenia (DN), determined by computing the number of days ANC was below the ANC of untreated control group during the recovery period.
  • the data are mean values from 5 animals.
  • the error bars represent SEM values. **p ⁇ 0.01; ***p ⁇ 0.001; ****p ⁇ 0.0001.
  • FIGS. 7A, 7B, 7C, 7D, 7E, and 7F are a set of graphs showing efficacy in neutropenic rats following administration of Eflapegrastim and Pegfilgrastim concomitantly and at different times up to 24 hours after docetaxel-CPA (TC) chemotherapy. Rats were administered with docetaxel (4 mg /kg) and CPA (32 mg/kg) to induce neutropenia and treated with Eflapegrastim or Pegfilgrastim at 0, 2, 5, and 24 hours after chemotherapy. Blood samples were collected via jugular vein up to 8 days. FIGS.
  • FIG. 7A, 7B, 7C, and 7D show ANC profiles following administration of Eflapegrastim or Pegfilgrastim at 0, 2, 5, and 24 hours after chemotherapy, respectively.
  • FIG. 7E shows area under the ANC vs. Time curve above baseline (AUECANC).
  • FIG. 7F shows the duration of neutropenia (DN), determined by computing the number of days ANC was below the ANC of untreated control group during the recovery period. The data are mean values from 5 animals. The error bars represent SEM values. **p ⁇ 0.01; ***p ⁇ 0.001; ****p ⁇ 0.0001
  • Chemotherapy may cause myelosuppression, potentially reducing bone marrow stem cell proliferation and subsequently decreased absolute neutrophil count (ANC).
  • ANC absolute neutrophil count
  • this disclosure provides a method of preventing, alleviating or treating a condition in a subject in need thereof, wherein the condition is characterized by compromised white blood cell production in the subject.
  • the method comprises administering to the subject a therapeutically effective amount of a chemotherapeutic regimen followed by a therapeutically effective amount of a protein complex comprising a modified human granulocyte-colony stimulating factor (hG-CSF) covalently linked to an immunoglobulin Fc region via a non-peptidyl polymer, wherein the non-peptidyl polymer is site-specifically linked to an N-terminus of the immunoglobulin Fc region, wherein the modified hG-CSF comprises substitutions at positions Cysl7 and Pro65, and wherein the protein complex is administered on the same day as the chemotherapeutic regimen.
  • hG-CSF human granulocyte-colony stimulating factor
  • this disclosure provides a method for increasing stem cell production in a donor.
  • the method comprises administering to the subject a therapeutically effective amount of a chemotherapeutic regimen followed by a therapeutically effective amount of a protein complex comprising a modified hG-CSF covalently linked to an immunoglobulin Fc region via a non- peptidyl polymer, wherein the non-peptidyl polymer is site-specifically linked to an N-terminus of the immunoglobulin Fc region, wherein the modified hG-CSF comprises substitutions at positions Cysl7 and Pro65, and wherein the protein complex is administered on the same day as the chemotherapeutic regimen.
  • this disclosure provides a method for increasing the number of granulocytes in eligible patients for a bone marrow transplant.
  • the method comprises administering to the subject a therapeutically effective amount of a chemotherapeutic regimen followed by a therapeutically effective amount of a protein complex comprising a modified hG- CSF covalently linked to an immunoglobulin Fc region via a non-peptidyl polymer, wherein the non-peptidyl polymer is site-specifically linked to an N-terminus of the immunoglobulin Fc region, wherein the modified hG-CSF comprises substitutions at positions Cysl7 and Pro65, and wherein the protein complex is administered on the same day as the chemotherapeutic regimen.
  • the present invention is directed to increasing the number of hematopoietic progenitor cells in a patient undergoing chemotherapy or in a patient who is a donor of a stem cell donor to a patient.
  • the method comprises administering to the subject a therapeutically effective amount of a chemotherapeutic regimen followed by a therapeutically effective amount of a protein complex comprising a modified hG-CSF covalently linked to an immunoglobulin Fc region via a non-peptidyl polymer, wherein the non-peptidyl polymer is site- specifically linked to an N-terminus of the immunoglobulin Fc region, wherein the modified hG- CSF comprises substitutions at positions Cysl7 and Pro65, and wherein the protein complex is administered on the same day as the chemotherapeutic regimen.
  • the conditions to be treated include reduced hematopoietic function, reduced immune function, reduced neutrophil count, reduced neutrophil mobilization, mobilization of peripheral blood progenitor cells, sepsis, severe chronic neutropenia, bone marrow transplants, infectious diseases, leucopenia, thrombocytopenia, anemia, enhancing engraftment of bone marrow during transplantation, enhancing bone marrow recovery in treatment of radiation, chemical or chemotherapeutic induced bone marrow aplasia or myelosuppression, and acquired immune deficiency syndrome.
  • the condition is myelosuppression, neutropenia, or preferably febrile neutropenia.
  • this disclosure provides a method for preventing, alleviating, prophylactically treating, and treating an infection as manifested by neutropenia (e.g., febrile neutropenia in the subject with non-myeloid malignancies receiving myelosuppressive anti-cancer drugs.
  • neutropenia e.g., febrile neutropenia in the subject with non-myeloid malignancies receiving myelosuppressive anti-cancer drugs.
  • the method comprises administering to the subject a therapeutically effective amount of a chemotherapeutic regimen followed by a therapeutically effective amount of a protein complex comprising a modified hG-CSF covalently linked to an immunoglobulin Fc region via a non- peptidyl polymer, wherein the non-peptidyl polymer is site-specifically linked to an N-terminus of the immunoglobulin Fc region, wherein the modified hG-CSF comprises substitutions at positions Cysl7 and Pro65, and wherein the protein complex is administered on the same day as the chemotherapeutic regimen.
  • the compromised white blood cell production is a result of chemotherapy, radiation therapy, adjuvant or neoadjuvant chemotherapy, or idiopathic thrombocytopenic purpura.
  • the adjuvant or neoadjuvant chemotherapy is a combination of docetaxel and cyclophosphamide.
  • the therapeutically effective amount is a unit dosage between about 5 pg/kg and about 200 pg/kg. In some embodiments, the therapeutically effective amount is a unit dosage form selected from: about 9 pg/kg, about 25 pg/kg, about 26 pg/kg, about 50 pg/kg, about 52 pg/kg, about 88 pg/kg, about 100 pg/kg, and about 200 pg/kg. In some embodiments, the present methodology further includes administering to the subject a therapeutically effective amount of a second agent, such as an anti-cancer agent.
  • the modified G-CSF is by way of a substitution at Cysl7 is Cysl7Ser. In other embodiments, the substitution at Pro65 is Pro65Ser. In some embodiments, the substitution is both the substitution Cysl7Ser and Pro65Ser may be referred herein as Ser 17, 55 .
  • the immunoglobulin Fc region comprises a polypeptide sequence of SEQ ID NO: 1.
  • the modified G-CSF comprises a polypeptide sequence of SEQ ID NO: 2.
  • the protein complex employed in the present methods contain (a) each domain of the immunoglobulin Fc fragment is a hybrid of domains, in which each domain has a different origin derived from immunoglobulins selected from IgG, IgA, IgD, IgE, and IgM; (b) the immunoglobulin Fc fragment is a dimer or multimer consisting of single-chain immunoglobulins comprising domains having the same origin; (c) the immunoglobulin Fc fragment is an IgG4 Fc fragment; or (d) the immunoglobulin Fc fragment is a human aglycosylated IgG4 Fc fragment.
  • the non-peptidyl polymer is selected from polyethylene glycol, polypropylene glycol, an ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, a biodegradable polymer, a lipid polymer, chitin, hyaluronic acid, and a combination thereof.
  • the non- peptidyl polymer is polyethylene glycol.
  • Another aspect of the present invention is directed to methods for treating or preventing neutropenia in a patient receiving chemotherapy comprising administering to said patient a protein complex comprising a modified G-CSF linked to an immunoglobulin Fc region via a non-peptidyl polymer, wherein the non-peptidyl polymer is site-specifically linked to an N-terminus of the immunoglobulin Fc region.
  • both ends of the non-peptidyl polymer are respectively linked to the physiologically active polypeptide and the immunoglobulin Fc region through reactive groups by a covalent bond.
  • the immunoglobulin Fc region is aglycosylated.
  • the protein complex is administered on the same day as the chemotherapeutic agent.
  • the G-CSF complex composition is administered to the patient within about 26, 24, 18, 12, 8, 6, 5, 3, 2, 1, or half-hour of the completion of chemotherapy. In some embodiments, the G-CSF complex is administered concurrently with the chemotherapy.
  • the G-CSF complex is a Ser 17 , 65 -GCSF-polyethylene glycol-IgG4- Fc, which is the conjugate of a recombinant human GCSF analog and human IgG4-Fc fragment connected via two chemical bonds between an amino group of N-terminus in each protein and one molecule of polyethylene glycol dialdehyde.
  • the G-CSF complex is a 17,65S G-CSF-PEG-FC protein complex.
  • the present invention is directed to a method for treating or preventing neutropenia in a patient diagnosed with cancer selected from non-small cell lung cancer, breast cancer, gastric cancer, colon cancer, pancreatic cancer, prostate cancer, myeloma, head and neck cancer, ovarian cancer, esophageal cancer, and metastatic cell carcinoma, comprising administering a chemotherapy regimen and a protein complex at the same day wherein the protein complex is administered within about 26, 24, 22, 18, 12, 8, 6, 5, 3, 2, 1, or half hour of the completion of chemotherapy.
  • cancer selected from non-small cell lung cancer, breast cancer, gastric cancer, colon cancer, pancreatic cancer, prostate cancer, myeloma, head and neck cancer, ovarian cancer, esophageal cancer, and metastatic cell carcinoma
  • the present invention is directed to a method for treating or preventing neutropenia in a patient diagnosed with breast cancer comprising administering a chemotherapy regimen of docetaxel and cyclophosphamide and therapeutically effective amounts o f 17 65S G-CSF-PEG-FC protein complex at doses of about 13.2 mg/0.6 mL (3.6 mg G-CSF equivalent), wherein the protein complex is administered 30 minutes, 2 hours, 3 hours, 5 hours, 8 hours, or 12 hours from the end of docetaxel and cyclophosphamide administration.
  • the chemotherapy regimen consisted of 3, 4, 5 or 6 cycles of 21 days, wherein on Day 1 of each cycle: (i) Docetaxel was administered at 75 mg/m 2 IV infusion per institute’s standard of care (ii) Cyclophosphamide 600 mg/m 2 IV infusion.
  • the duration of neutropenia from the first occurrence of an ANC below the threshold is unexpectedly superior for 17,65S G-CSF-PEG-Fc protein complex (Eflapegrastim) in patients suffering from non-small cell lung cancer, breast cancer, gastric cancer, colon cancer, pancreatic cancer, prostate cancer, myeloma, head and neck cancer, ovarian cancer, esophageal cancer, and metastatic cell carcinoma, as compared to other G-CSF or analogs thereof.
  • such superior results may be observed in any of the treatment cycles, including but not limited to cycle 1, 2, 3 or 4.
  • the incidences of adverse events were substantially lower as measured by competent clinical assessments as compared to other G-CSF or analogs thereof.
  • the patient is diagnosed with breast cancer.
  • the duration of neutropenia is assessed based on the severity as the number of postdose days of severe neutropenia (ANC ⁇ 0.5x l0 9 /L) from the first occurrence of an ANC below the threshold.
  • the duration of neutropenia is unexpectedly superior for 17 65S G- CSF-PEG-Fc protein complex (Eflapegrastim) as compared to other G-CSF or analogs thereof in patients suffering from breast cancer, when at least one dose of 1765S G-CSF-PEG-Fc protein complex is administered about 24, 18, 12, 8, 6, 5, 3, 2, 1, or half-hour of the completion of chemotherapy. In some embodiments, 65S G-CSF-PEG-Fc protein complex is administered about 6, 5, 3, 2, 1, or half hour of the completion of chemotherapy.
  • the chemotherapy comprises therapeutically effective doses of docetaxol and cyclophosphamide
  • such superior results may be observed in any of the treatment cycles, including but not limited to cycle 1, 2, 3 or 4.
  • the incidences of adverse events were substantially lower as measured by competent clinical assessments as compared to other G-CSF or analogs thereof.
  • the immunoglobulin Fc region consists of one to four domains selected from CHI, CH2, CH3, and CH4 domains.
  • the immunoglobulin Fc region further includes a hinge region.
  • the present invention provides the protein complex in which the immunoglobulin Fc region is an immunoglobulin Fc fragment derived from IgG, IgA, IgD, IgE, or IgM.
  • the present invention provides the protein complex in which each domain of the immunoglobulin Fc fragment is a hybrid of domains, and each domain has a different origin derived from immunoglobulins selected from IgG, IgA, IgD, IgE, and IgM.
  • the immunoglobulin Fc fragment is a dimer or multimer consisting of single chain immunoglobulins comprising domains having the same origin.
  • the immunoglobulin Fc fragment is an IgG4 Fc fragment.
  • the present invention provides the protein complex in which the non-peptidyl polymer is selected from polyethylene glycol, polypropylene glycol, an ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, a biodegradable polymer, a lipid polymer, chitin, hyaluronic acid, and a combination thereof.
  • the non-peptidyl polymer is polyethylene glycol.
  • the non-peptidyl polymer is 3.4 kDa polyethylene glycol.
  • the reactive group of the non-peptidyl polymer is selected from an aldehyde group, a maleimide group, and a succinimide derivative.
  • the aldehyde group is a propionaldehyde group or a butyraldehyde group.
  • the succinimide derivative is succinimidyl carboxymethyl, succinimidyl valerate, succinimidyl methylbutanoate, succinimidyl methylpropionate, succinimidyl butanoate, succinimidyl propionate, N-hydroxysuccinimide, or succinimidyl carbonate.
  • the non-peptidyl polymer has an aldehyde group as a reactive group at both ends. In some embodiments, the non-peptidyl polymer has an aldehyde group and a maleimide group as a reactive group at both ends, respectively. In some embodiments, the non- peptidyl polymer has an aldehyde group and a succinimide group as a reactive group at both ends, respectively.
  • each end of the non-peptidyl polymer is linked to the N-terminus of the immunoglobulin Fc region; and the N-terminus, C-terminus, or a free reactive group of a lysine residue, a histidine residue, or a cysteine residue of the physiologically active polypeptide, respectively.
  • the physiologically active polypeptide is selected from a hormone, a cytokine, an enzyme, an antibody, a growth factor, a transcription factor, a blood coagulation factor, a vaccine, a structural protein, a ligand protein, and a receptor.
  • the protein complex is a Ser 17 ’ 65S -GCSF-polyethylene glycol-IgG4- Fc which is the conjugate of a recombinant human GCSF analog and human IgG4-Fc fragment connected via two chemical bonds between an amino group of N-terminus in each protein and one molecule of polyethylene glycol dialdehyde with the molecular weight ranging from 1 kDa to 200 kDa, preferably between 1 kDa to 100 kDa.
  • the molecular weight of the protein complex, including the GCSF analog, the IgG4-FC fragment, and the polyethylene glycol dialdehyde is 72 kDa.
  • protein complex or “complex” refers to a structure in which at least one physiologically active polypeptide, at least one non-peptidyl polymer having a reactive group at both ends thereof, and at least one immunoglobulin Fc region are linked to each other via a covalent bond. Further, a structure in which only two molecules selected from the physiologically active polypeptide, the non-peptidyl polymer, and the immunoglobulin Fc region are linked to each other via a covalent bond is called “conjugate” in order to distinguish it from the “complex.”
  • the protein complex of the present invention may be a protein complex in which the PEG is linked to the modified G-CSF and the immunoglobulin Fc region through reactive groups at both ends thereof by a covalent bond, respectively.
  • physiologically active polypeptide refers to a polypeptide or a protein having some kind of antagonistic activity to a physiological event in vivo, and these terms may be used interchangeably.
  • non-peptidyl polymer refers to a biocompatible polymer including two or more repeating units which are linked to each other by any covalent bond excluding a peptide bond, but is not limited thereto.
  • immunoglobulin Fc region refers to a region of an immunoglobulin molecule, except for the variable regions of the heavy and light chains, the heavychain constant region 1 (CHI) and the light-chain constant region 1 (CL1) of an immunoglobulin.
  • the immunoglobulin Fc region may further include a hinge region at the heavy-chain constant region.
  • the immunoglobulin Fc region of the present invention may be a fragment, including a part or all of the Fc region, and in the present invention, the immunoglobulin Fc region may be used interchangeably with an immunoglobulin fragment.
  • a native Fc has a sugar chain at position Asn297 of heavy-chain constant region 1, but A. co/z-derived recombinant Fc is expressed as an aglycosylated form.
  • the removal of sugar chains from Fc results in a decrease in binding affinity of Fc gamma receptors 1, 2, and 3 and complement (Clq) to heavy-chain constant region 1, leading to a decrease or loss in antibody-dependent cell- mediated cytotoxicity or complement-dependent cytotoxicity.
  • immunoglobulin constant region may refer to an Fc fragment including heavy-chain constant region 2 (CH2) and heavy-chain constant region 3 (CH3) (or containing heavy-chain constant region 4 (CH4)), except for the variable regions of the heavy and light chains, the heavy-chain constant region 1 (CHI) and the light-chain constant region (CL) of an immunoglobulin, and may further include a hinge region at the heavy chain constant region.
  • CH2 heavy-chain constant region 2
  • CH3 heavy-chain constant region 3
  • CH4 heavy-chain constant region 4
  • the immunoglobulin constant region of the present invention may be an extended immunoglobulin constant region including a part or all of the Fc region, including the heavy-chain constant region 1 (CHI) and/or the light chain constant region (CL), except for the variable regions of the heavy and light chains of an immunoglobulin, as long as it has a physiological function substantially similar to or better than the native protein.
  • CHI heavy-chain constant region 1
  • CL light chain constant region
  • the immunoglobulin constant region may originate from humans or animals, such as cows, goats, pigs, mice, rabbits, hamsters, rats, guinea pigs, etc., and may preferably be of human origin.
  • the immunoglobulin constant region may be selected from constant regions derived from IgG, IgA, IgD, IgE, IgM, or combinations or hybrids thereof, preferably, derived from IgG or IgM, which are the most abundant thereof in human blood, and most preferably, derived from IgG, which is known to improve the half-life of ligand-binding proteins.
  • the immunoglobulin Fc region may be a dimer or multimer consisting of single-chain immunoglobulins of domains of the same origin.
  • the term “combination” means that polypeptides encoding single-chain immunoglobulin constant regions (preferably Fc regions) of the same origin are linked to a singlechain polypeptide of a different origin to form a dimer or multimer. That is, a dimer or a multimer may be prepared from two or more fragments selected from Fc fragments of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE Fc.
  • hybrid means that sequences encoding two or more immunoglobulin constant regions of different origins are present in a single-chain of an immunoglobulin constant region (preferably, an Fc region).
  • the hybrid domain may be composed of one to four domains selected from CHI, CH2, CH3, and CH4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc, and IgD Fc, and may further include a hinge region.
  • IgG may be divided into the IgGl, IgG2, IgG3, and IgG4 subclasses, and the present invention may include combinations or hybrids thereof. Preferred are the IgG2 and IgG4 subclasses, and most preferred is the Fc region of IgG4 rarely having effector functions such as complement dependent cytotoxicity (CDC).
  • CDC complement dependent cytotoxicity
  • the immunoglobulin constant region may have the glycosylated form to the same extent as or to a greater or lesser extent than the native form or maybe the deglycosylated form. Increased or decreased glycosylation or deglycosylation of the immunoglobulin constant region may be achieved by typical methods, for example, by using a chemical method, an enzymatic method, or a genetic engineering method using microorganisms.
  • the complement (Clq) binding to an immunoglobulin constant region becomes significantly decreased, and antibody-dependent cytotoxicity or complement-dependent cytotoxicity is reduced or removed, thereby not inducing unnecessary immune responses in vivo.
  • the immunoglobulin Fc region may be more specifically an aglycosylated Fc region derived from human IgG4, that is, a human IgG4-derived aglycosylated Fc region.
  • the human-derived Fc region is more preferable than a non-human derived Fc region, which may act as an antigen in the human body and cause undesirable immune responses such as the production of a new antibody against the antigen.
  • the immunoglobulin constant region of the present invention includes not only the native amino acid sequence but also sequence derivatives (mutants) thereof.
  • the amino acid sequence derivative means that it has an amino acid sequence different from the wild-type amino acid sequence as a result of deletion, insertion, conserved or non-conserved substitution of one or more amino acid residues, or a combination thereof. For instance, amino acid residues at positions 214 to 238, 297 to 299, 318 to 322, or 327 to 331 in IgG Fc, known to be important for linkage, may be used as the sites suitable for modification.
  • Various derivatives such as those prepared by removing the sites capable of forming disulfide bonds, removing several N-terminal amino acids from native Fc, or adding methionine to the N-terminus of native Fc, may be used.
  • complement fixation sites e.g., Clq fixation sites, or ADCC sites, may be eliminated to remove the effector function.
  • the techniques of preparing the sequence derivatives of the immunoglobulin constant region are disclosed in International Patent Publication Nos. WO 97/34631 and WO 96/32478.
  • amino acids may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, famesylation, acetylation, amidation, or the like.
  • the above-described immunoglobulin constant region derivative may be a derivative which has a biological activity equivalent to that of the immunoglobulin constant region, but has increased structural stability of the immunoglobulin constant region against heat, pH, etc.
  • the immunoglobulin constant region may be obtained from a native type isolated from humans or animals such as cows, goats, pigs, mice, rabbits, hamsters, rats, guinea pigs, etc., or may be their recombinants or derivatives obtained from transformed animal cells or microorganisms.
  • they may be obtained from a native immunoglobulin by isolating whole immunoglobulins from human or animal organisms and treating them with a proteolytic enzyme.
  • a human-derived immunoglobulin constant region may be a recombinant immunoglobulin constant region that is obtained from a microorganism.
  • the protein complex of the present disclosure may include one or more of a unit structure of a [physiologically active polypeptide/non-peptidyl polymer/immunoglobulin Fc region], in which all components may be linked in a linear form by a covalent bond.
  • the non-peptidyl polymer may have a reactive group at both ends thereof and is linked to the physiologically active polypeptide and the immunoglobulin Fc region through the reactive group by a covalent bond, respectively.
  • At least one conjugate of the physiologically active polypeptide and the non- peptidyl polymer is linked to one immunoglobulin Fc region by a covalent bond, thereby forming a monomer, dimer, or multimer of the physiologically active polypeptide, which is mediated by the immunoglobulin Fc region. Therefore, an increase in vivo activity and stability may be more effectively achieved.
  • the reactive group at both ends of the non-peptidyl polymer is preferably selected from a reactive aldehyde group, a propionaldehyde group, a butyraldehyde group, a mal eimide group, and a succinimide derivative.
  • the succinimide derivative may be hydroxy succinimidyl, succinimidyl carboxymethyl, succinimidyl valerate, succinimidyl methyl butanoate, succinimidyl methyl propionate, succinimidyl butanoate, succinimidyl propionate, N-hydroxysuccinimide, or succinimidyl carbonate.
  • non-peptidyl polymer when the non-peptidyl polymer has a reactive aldehyde group at both ends, it is effective in linking both of the ends with the physiologically active polypeptide and the immunoglobulin with minimal non-specific reactions.
  • a final product generated by reductive alkylation by an aldehyde bond is much more stable than when linked by an amide bond.
  • the reactive groups at both ends of the non-peptidyl polymer of the present invention may be the same as or different from each other.
  • the non-peptide polymer may possess aldehyde reactive groups at both ends, or it may possess an aldehyde group at one end and a maleimide reactive group at the other end, or an aldehyde group at one end and a succinimide reactive group at the other end, but is not limited thereto.
  • the non-peptide polymer may possess a maleimide group at one end and an aldehyde group, a propionaldehyde group, or a butyraldehyde group at the other end.
  • the non-peptide polymer may possess a succinimidyl group at one end and a propionaldehyde group, or a butyraldehyde group at the other end.
  • a polyethylene glycol having a reactive hydroxy group at both ends thereof is used as the non-peptidyl polymer, the hydroxy group may be activated to various reactive groups by known chemical reactions, or a commercially available polyethylene glycol having a modified reactive group may be used so as to prepare the protein complex of the present invention.
  • each of both of the ends of the non-peptidyl polymer may bind to the N-terminus of the immunoglobulin Fc region and theN-terminus (amino terminus), C-terminus (carboxy terminus), or free reactive group of a lysine residue, a histidine residue, or a cysteine residue of the physiologically active polypeptide.
  • N-terminus refers to an N-terminus of a peptide, which is a site to which a linker including a non-peptidyl polymer can be conjugated for the purpose of the present invention.
  • Examples of the N-terminus may include not only amino acid residues at the distal end of the N-terminus, but hut also amino acid residues near the N-terminus, but are not limited thereto. Specifically, the 1st to the 20th amino acid residues from the distal end may be included.
  • the non-peptidyl polymer of the present invention may be selected from polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, biodegradable polymers such as PLA (polylactic acid) and PLGA (polylactic-glycolic acid), lipid polymers, chitins, hyaluronic acid, and combinations thereof, and specifically, polyethylene glycol, but is not limited thereto. Also, derivatives thereof well known in the art and easily prepared within the skill of the art are included in the non-peptidyl polymer of the present invention.
  • the non-peptidyl polymer may have a molecular weight in the range of 1 kDa to 100 kDa, and specifically 1 kDa to 20 kDa.
  • physiologically active polypeptide may be exemplified by various physiologically active polypeptides such as hormones, cytokines, interleukins, interleukin-binding proteins, enzymes, antibodies, growth factors, transcription factors, blood factors, vaccines, structural proteins, ligand proteins or receptors, cell surface antigens, receptor antagonists, and derivatives or analogs thereof.
  • physiologically active polypeptide includes human growth hormones, growth hormone-releasing hormones, growth hormone-releasing peptides, interferons and interferon receptors (e.
  • interferon-alpha, -beta, and -gamma, soluble type I interferon receptors colony-stimulating factors
  • interleukins e.g., interleukin- 1, -2, -3, -4, -6, -7, -8, -9, -10, -11, -12, -13, -14, -15, -16, -17, -18, -19,. -20, -21, -22, -23, -24, -25, -26, -27, -28., - 29. -30.
  • interleukin receptors e.g;.. IL-1 receptor.
  • IL-4 receptor etc.
  • enzymes e.g., glucocerebrosidase, iduronate-2-sulfatase, alpha- galactosidase- A, agalsidase alpha, beta, alpha-L- iduronidase, butyrylcholinesterase, chitinase, glutamate decarboxylase, imiglucerase, lipase, uricase, platelet-activating factor acetylhydrolase, neutral endopeptidase, myeloperoxidase, etc ), interleukin- and cytokine-binding proteins e.g., IL-18bp, TNF -binding protein, etc.), macrophageactivating factors, macrophage peptides, B-cell factors, T-cell factors, protein A, allergy inhibitors, cell necrosis glycoproteins, immunotoxins, lymphotoxins, tumor necrosis factor, tumor suppressors, transforming growth factor, alpha
  • the antibody fragment may be Fab, Fab', F(ab'), Fd, or scFv having an ability to bind to a specific antigen, and preferably, Fab.
  • the Fab fragments include the variable domain (VL) and constant domain (CL) of the light chain and the variable domain (VH) and the first constant domain (CHI) of the heavy chain.
  • the Fab' fragments differ from the Fab fragments in terms of the addition of several amino acid residues including one or more cysteine residues from the hinge region at the carboxyl terminus of the CHI domain.
  • the Fd fragments are fragments consisting of only the VH and CHI domains, and the F(ab')2 fragments are produced by binding of two molecules of Fab' fragments by either disulfide bonding or a chemical reaction.
  • the scFv fragment is a single polypeptide chain, in which only VL and VH domains are linked to each other by a peptide linker.
  • the protein complex of the present invention may be used in the development of long-acting protein formulations of animal growth hormone such as bovine growth hormone or porcine growth hormone, and long-acting protein formulations for treatment or prevention of animal disease, such as interferon.
  • the preferred protein complex according to the present invention is a 17,65S G-CSF-polyethylene glycol-IgG4-Fc which is the conjugate of a recombinant human G-CSF analog and human IgG4-Fc fragment connected via two chemical bonds between an amino group of N-terminus in each protein and one molecule of polyethylene glycol dialdehyde having the molecular weight of 72 kDa.
  • the protein complex as disclosed can be prepared by a method comprising: (a) preparing a protein complex by linking at least one non-peptidyl polymer having a reactive group at both ends, at least one physiologically active polypeptide, and at least one immunoglobulin Fc region by a covalent bond, and (b) isolating the protein complex, essentially including the covalently linked physiologically active polypeptide, non-peptidyl polymer, and immunoglobulin Fc region prepared in step (a), in which the non-peptidyl polymer is linked to the N-terminus of the immunoglobulin Fc fragment.
  • the immunoglobulin Fc region of the present invention may be in the form of a dimer, or in the form of a homodimer or heterodimer.
  • the immunoglobulin Fc region constituting the protein complex of the present invention may include one or two or more of an N-terminus.
  • the immunoglobulin Fc region may be linked to at least one non-peptidyl polymer via the N-terminus.
  • the immunoglobulin Fc region of the present invention may be in the form of a homodimer, and therefore, linked to one or two non-peptidyl polymers via two N- terminals included in the homodimer of the immunoglobulin Fc region.
  • the non- peptidyl polymers may bind to the physiologically active polypeptides, respectively, thereby forming the protein complex.
  • the protein complex of the present invention may be prepared by linking one or two or more of the non-peptidyl polymer, one or two or more of the physiologically active polypeptide, and one or two or more of the immunoglobulin Fc region via a covalent bond.
  • step (a) the covalent bonds between the three components may occur sequentially or at the same time.
  • any one of the physiologically active polypeptide and the immunoglobulin Fc region may be first linked to one end of the non-peptidyl polymer, and then the other may be linked to the other end of the non- peptidyl polymer. This method is advantageous in that production of by-products other than the desired protein complex is minimized, and the protein complex is prepared in high purity.
  • step (a) may comprise:
  • step (iii) producing a protein complex by linking the physiologically active polypeptide or the specific site of the immunoglobulin Fc region to the other end of the non-peptidyl polymer of the isolated conjugate.
  • step (a) includes (al) preparing a conjugate by linking one end of the non-peptidyl polymer to any one of the immunoglobulin Fc region and the physiologically active polypeptide by a covalent bond; and (a2) isolating the conjugate prepared in step (al) and linking the other end of the non-peptidyl polymer of the isolated conjugate to the other of the physiologically active polypeptide and the immunoglobulin Fc region by a covalent bond.
  • step (a) may comprise (af) preparing a conjugate by linking one end of the non-peptidyl polymer to the immunoglobulin Fc region by a covalent bond; and (a2') isolating the conjugate prepared in step (af) and linking the other end of the non-peptidyl polymer of the isolated conjugate to the physiologically active polypeptide by a covalent bond.
  • step (a) may include (al”) preparing a conjugate by linking one end of the non-peptidyl polymer to the physiologically active polypeptide by a covalent bond; and (a2”) isolating the conjugate prepared in step (al”) and linking the other end of the non-peptidyl polymer of the isolated conjugate to the immunoglobulin Fc region by a covalent bond.
  • reaction mole ratio between the physiologically active polypeptide and the non-peptidyl polymer may be in the range from 1 : 1 to 1 :30, and the reaction mole ratio between the immunoglobulin Fc region and the non-peptidyl polymer may be in the range from 1 : 1 to 1 :20.
  • the reaction mole ratio between the immunoglobulin Fc region and the non-peptidyl polymer may be in the range from 1 : 1 to 1 :20, and in particular, in the range from 1 :1 to 1 :15, 1 :1 to 1 :10, or 1 :1 to 1 :4.
  • the reaction mole ratio between the physiologically active polypeptide and the non-peptidyl polymer may be in the range from 1 : 1 to 1 :30, and in particular, in the range from 1 :1 to 1:15 or 1: 1 to 1 : 10.
  • a preparation yield and cost may be optimized depending on the reaction mole ratio.
  • step (al), (al), or (al”) may be performed in a pH condition from 4.0 to 9.0; step (al), (al 1 ), or (al”) may be performed at a temperature from 4.0°C to 25°C; in step (al), (al 1 ), or (al”), the reaction concentration of the immunoglobulin Fc region or physiologically active polypeptide may be in the range from 0.1 mg/mL to 100 mg/mL.
  • the reaction mole ratio between the conjugate and the immunoglobulin Fc region or the physiologically active polypeptide may be in the range from 1 :0.1 to 1 :20, and in particular, in the range from 1 :0.2 to 1 : 10.
  • the reaction mole ratio between the conjugate and the physiologically active polypeptide may be in the range from 1 :0.1 to 1 :20
  • the reaction mole ratio between the conjugate and the immunoglobulin Fc region may be in the range from 1 :0.1 to 1 :20.
  • a preparation yield and cost may be optimized depending on the reaction mole ratio.
  • step (a2), (a2'), or (a2”) may be performed in a pH condition from 4.0 to 9.0; step (a2), (a2'), or (a2”) may be performed at a temperature from 4.0°C to 25°C; in step (a2), (a2'), or (a2”), the reaction concentration of the immunoglobulin Fc region or physiologically active polypeptide may be in the range from 0.1 mg/mL to 100 mg/mL.
  • the preparation method of the present invention may be a method of preparing a position-specific protein complex, including (a 1 ) preparing a conjugate by linking one end of the non-peptidyl polymer to any one of the immunoglobulin Fc region and the physiologically active polypeptide by a covalent bond, in which the reaction mole ratio between the physiologically active polypeptide and the non-peptidyl polymer is in the range from 1 : 1 to 1 :30, the reaction mole ratio between the immunoglobulin Fc region and the non-peptidyl polymer is in the range from 1 :1 to 1 :20, a reducing agent is contained in the range from 1 mM to 100 mM, the reaction is performed in the condition of pH from 4.0 to 9.0, at a temperature from 4.0°C to 25°C, and the reaction concentration of the immunoglobulin Fc region or physiologically active polypeptide is in the range from 0.1 mg/mL to 100 mg/mL;
  • step (b 1 ) isolating the conjugate prepared in step (a 1 ) and linking the other end of the non- peptidyl polymer of the isolated conjugate to the other of the immunoglobulin Fc region and the physiologically active polypeptide by a covalent bond, in which the reaction mole ratio between the conjugate and the immunoglobulin Fc region or the physiologically active polypeptide is in the range from 1 :0.1 to 1:20, a reducing agent is contained in the range from 1 mM to 100 mM, the reaction is performed in the condition of pH from 4.0 to 9.0, at a temperature from 0°C to 25°C, and the concentration of the immunoglobulin Fc region or physiologically active polypeptide is in the range from 0.1 mg/mL to 100 mg/mL; and (o') isolating the protein complex, essentially including the covalently linked physiologically active polypeptide, non-peptidyl polymer, and immunoglobulin Fc region prepared in step (b 1 ), in which the non
  • step (al), step (af), step (al”), step (a2), step (a2'), and step (a2”) of the present invention may be performed in the presence of a reducing agent, considering the type of the reactive groups at both ends of the non-peptidyl polymer which participate in the reactions, if necessary.
  • the reducing agent of the present invention may be sodium cyanoborohydride (NaCNBH3), sodium borohydride, dimethylamine borate, or pyridine borate.
  • a concentration of the reducing agent e.g., sodium cyanoborohydride
  • temperature and pH of a reaction solution e.g., sodium cyanoborohydride
  • total concentrations of the physiologically active polypeptide and the immunoglobulin Fc region participating in the reaction are important in terms of production yield and purity.
  • various combinations of the conditions are needed.
  • the physiologically active polypeptide to be prepared various conditions are possible, but not limited to, the reducing agent (e.g., sodium cyanoborohydride) may be contained in the range from 1 mM to 100 mM, the reaction solution may be at a temperature from 0°C to 25°C and in the condition of pH from 4.0 to 9.0, and the concentration of the reaction protein (concentration of the immunoglobulin Fc region or physiologically active polypeptide included upon the reaction) may be in the range from 5 mg/mL to 100 mg/mL.
  • the reducing agent e.g., sodium cyanoborohydride
  • the reaction solution may be at a temperature from 0°C to 25°C and in the condition of pH from 4.0 to 9.0
  • concentration of the reaction protein concentration of the immunoglobulin Fc region or physiologically active polypeptide included upon the reaction
  • the separation of the conjugate in step (a2), step (a2'), and step (a2”) may be performed, if necessary, by a method selected from general methods which are used in protein separation, considering the properties such as purity, hydrophobicity, molecular weight, and electrical charge which are required for the separated conjugate.
  • the separation may be performed by applying various known methods, including size exclusion chromatography, affinity chromatography, hydrophobic chromatography, or ion exchange chromatography, and if necessary, a plurality of different methods are used in combination to purify the conjugate with higher purity.
  • physiologically active polypeptide to be prepared various conditions are possible.
  • size exclusion chromatography is generally performed.
  • affinity chromatography, hydrophobic chromatography, or ion exchange chromatography may also be used.
  • Step (b) may be performed, if necessary, by a method selected from general methods which are used in protein separation, considering the properties such as hydrophobicity, molecular weight, and electrical charge, in order to finally purify a high-purity complex.
  • the separation may be performed by applying various known methods, including size exclusion chromatography, affinity chromatography, hydrophobic chromatography, or ion exchange chromatography, and if necessary, a plurality of different methods are used in combination to purify the complex with higher purity.
  • various separation conditions are possible.
  • hydrophobic chromatography and ion exchange chromatography may be used in combination, and a plurality of hydrophobic chromatography or a plurality of ion exchange chromatography is also possible.
  • ion exchange chromatography or hydrophobic chromatography may be used singly.
  • the ion exchange chromatography is to separate a protein by passing charged protein at a specific pH through a charged ion resin-immobilized chromatography column and separating the protein by a difference in the migration rate of the protein, and it may be anion exchange chromatography or cation exchange chromatography.
  • the anion exchange chromatography is to use a cation resin, and a functional group of the resin constituting the corresponding anion exchange chromatography may be any one selected from quaternary ammonium (Q), quaternary aminoethyl (QAE), di ethyl aminoethyl (DEAE), polyethylene amine (PEI), dimethyl-laminomethyl (DMAE), and trimethylaminoethyl (TMAE), but is not limited thereto.
  • Q quaternary ammonium
  • Q quaternary aminoethyl
  • DAE di ethyl aminoethyl
  • PEI polyethylene amine
  • DMAE dimethyl-laminomethyl
  • TMAE trimethylaminoethyl
  • the cation exchange chromatography is to use an anion resin, and a functional group of the resin constituting the corresponding cation exchange chromatography may be any one selected from methyl sulfonate (S), sulfopropyl (SP), carboxymethyl (CM), sulfoethyl (SE), and polyaspartic acid, but is not limited thereto.
  • S methyl sulfonate
  • SP sulfopropyl
  • CM carboxymethyl
  • SE sulfoethyl
  • polyaspartic acid but is not limited thereto.
  • a functional group of the resin constituting the hydrophobic chromatography may be any one selected from phenyl, octyl, (iso)propyl, butyl, and ethyl, but is not limited thereto.
  • a functional group of the resin constituting the size exclusion chromatography may be any one selected from Superdex, Sephacryl, Superpose, and Sephadex, but is not limited thereto.
  • the affinity chromatography in the present invention is to separate a protein by a difference in the migration rate of the protein, which is caused by the interaction between the protein and a ligand capable of interacting with the protein in a resin onto which the ligand is immobilized.
  • a functional group of the resin constituting the affinity chromatography may be any one selected from protein A, heparin, blue, benzamidine, metal ions (cobalt, nickel, and copper), and an antibody to a part or the entirety of the constituting components of the protein complex, in which both ends of the non-peptidyl polymer arc respectively conjugated to the immunoglobulin Fc region and the physiologically active polypeptide, but is not limited thereto.
  • Step (b) is to isolate the protein complex in which the non-peptidyl polymer and the immunoglobulin Fc region are linked to each other via the N-terminus of the immunoglobulin Fc region.
  • Still another aspect of the present invention provides a method for preparing a protein complex with N-terminal selectivity of 90% or higher.
  • the protein complex prepared by the method of the present invention may be one, in which one end of the non-peptidyl polymer may be linked to the N-terminus of the immunoglobulin Fc region with N-terminal selectivity of 90% or higher, more specifically 95% or higher, even more specifically 98% or higher, and yet even more specifically 99% or higher, but is not limited thereto.
  • the term “linking with N-terminal selectivity of 90% or higher” means that, in 90% or more of the protein complex prepared by purification of the protein complex fractions obtained by a series of reactions according to the present invention, the non-peptidyl polymer is linked to the N-terminus of the Fc region in a position-specific manner.
  • the term “90% or higher” may refer to v/v, w/w, and peak/peak, but is not limited to a particular unit.
  • the yield of the protein complex comprising the non-peptidyl polymer linked to the N-terminus of the Fc region in a position-specific manner may vary by reaction conditions, a reactor of the non- peptidyl polymer, etc.
  • a protein complex with N- terminal selectivity of 90% or higher can be prepared by the method according to the present invention, via preparation of various physiologically active polypeptides, non-peptidyl polymers, and Fc complexes.
  • the pharmaceutical composition may comprise a protein complex, which includes the physiologically active polypeptide-non-peptidyl polymer-N-terminus of an immunoglobulin Fc region, in an amount of 90 % or higher, more specifically 95 % or higher, even more specifically 98 % or higher, and yet even more specifically 99 % or higher, but is not limited thereto.
  • the term “90% or higher” may refer to v/v, w/w, and peak/peak, but is not limited to a particular unit.
  • the pharmaceutical composition may further include a pharmaceutically acceptable excipient.
  • the pharmaceutical composition of the present invention may be administered via various routes, including oral, percutaneous, subcutaneous, intravenous, and intramuscular routes, e.g., in the form of an injectable formulation.
  • the pharmaceutical composition of the present invention may be formulated by a method known in the art in order to provide rapid, long-lasting, or delayed release of the active ingredient after administration thereof to a mammal.
  • the formulation may be a tablet, a pill, a powder, a sachet, an elixir, a suspension, an emulsion, a solution, a syrup, an aerosol, a soft or hard gelatin capsule, a sterile injectable solution, or a sterile powder.
  • suitable carriers, excipients, and diluents may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.
  • the pharmaceutical composition may further include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, a preservative, etc.
  • a practical administration dose of the protein complex of the present invention may be determined by several related factors, including the types of diseases to be treated, administration routes, the patient's age, gender, weight, and severity of the illness, as well as by the types of the physiologically active polypeptide as an active component. Since the protein complex of the present invention has excellent blood duration and in vivo potency, it can remarkably reduce the administration dose and frequency of a peptide drug, including the protein complex of the present invention.
  • the terms “population of complex” and “population” may be used interchangeably, and they refer to a group of protein complexes, including protein complexes, in which a non-peptidyl polymer is linked to the N-terminus of an Fc region, and/or protein complexes, in which a non-peptidyl polymer is linked to a region other than the N-terminus of an Fc region.
  • the population may include only the protein complexes, in which a non-peptidyl polymer is linked to the N-terminus of an Fc region, or the protein complexes, in which a non- peptidyl polymer is linked to a region other than the N-terminus of an Fc region.
  • the percentage of the protein complexes, in which a non-peptidyl polymer is linked to a region other than the N-terminus of an Fc region, included in the population may be 90% or higher, more specifically 95% or higher, even more specifically 98% or higher, and yet even more specifically 99% or higher, but is not limited thereto.
  • the term “90% or higher” may refer to v/v, w/w, and peak/peak, but is not limited to a particular unit.
  • the population may refer to a population with an increased percentage of the protein complexes, in which a non-peptidyl polymer is linked to a region other than the N-terminus of an Fc region, by removing impurities, unreacted materials, etc., from the protein complexes prepared thereof. Additionally, the population may refer to one which was prepared by a method for preparing protein complexes with N-terminal selectivity of 90% or higher, but is not limited thereto. The population may be efficiently purified by the method of the present invention.
  • the present invention is particularly directed to the use of the above-described protein complexes in preventing, alleviating, or treating patient in need thereof having a need in increasing their white blood cell production, count, or are in need of increasing stem cell production by administering to the patient a therapeutically effective amount of a protein complex comprising a modified human granulocyte-colony stimulating factor (hG-CSF) covalently linked to an immunoglobulin Fc region via a non-peptidyl polymer, wherein the non-peptidyl polymer is site- specifically linked to an N-terminus of the immunoglobulin Fc region, and the modified hG-CSF comprises substitutions in at least one of Cysl7 and Pro65.
  • hG-CSF human granulocyte-colony stimulating factor
  • Such methodologies may or may not be in combination with chemotherapeutic agents or regimens including docetaxel, doxorubicin, cyclophosphamide (TAC); dose-dense doxorubicin plus cyclophosphamide (AC), with or without subsequent weekly or semiweekly paclitaxel; and docetaxel plus cyclophosphamide (TC).
  • TAC docetaxel
  • AC dose-dense doxorubicin plus cyclophosphamide
  • TC docetaxel plus cyclophosphamide
  • At least one dose of Eflapegrastim is administered at 13.2 mg/0.6 mL (containing 3.6 mg G-CSF) fixed-dose to the patient within about 26 hours, 24 hours, 22 hours, 18 hours, 12 hours, 6 hours, about 5 hours, 2 hours, 1 hour or half an hour of the completion of chemotherapy.
  • TC is administered on Day 1 of each cycle intravenously (IV).
  • Docetaxel is administered at 75 mg/m 2 IV infusion and (ii) Cyclophosphamide is administered at 600 mg/m 2 IV infusion.
  • Each treatment cycle is 21 days, with up to a maximum of 4 cycles of chemotherapy.
  • Eflapegrastim may be administered on Day 2 of each cycle, approximately 24 hours ( ⁇ 2 hours) after TC chemotherapy.
  • ALD-PEG-ALD (IDB, Korea), which is polyethylene glycol (PEG) having a molecular weight of 3.4 kDa and aldehyde reactive groups at both ends thereof, was added to 5 mg/mL of human interferon alpha-2b (hIFNa-2b, molecular weight: 19 kDa) dissolved in 100 mM phosphate buffer at a molar ratio of hIFNa:PEG of 1:5 to 1 : 10.
  • a reducing agent, sodium cyanoborohydride (NaCNBHs, Sigma) was added thereto at a final concentration of 20 mM and allowed to react at 4°C to 8°C under slow stirring for about 1 hour.
  • reaction mixture was subj ected to SP HP (GE Healthcare) anion exchange chromatography to purify an IFNa-PEG conjugate with high purity.
  • the immunoglobulin Fc fragment was added and reacted at a molar ratio of IFNa-PEG conjugate: immunoglobulin Fc of 1 : 1 to 1 :4.
  • the reaction solution was prepared as 100 mM phosphate buffer (pH 5.5 to 6.5), and sodium cyanoborohydride (NaCNBH-, Sigma) was added as a reducing agent at a final concentration of 20 mM to 50 mM.
  • the reaction was allowed at 4°C to 8°C for about 12 hours to 16 hours under slow stirring.
  • the reaction mixture was buffer-exchanged to 10 mM Tris (pH 7.5) and then passed through a Source Q (GE Healthcare) anion exchange chromatography column to remove unreacted Fc and to obtain an IFNa-PEG-Fc protein complex fraction.
  • the reaction solution was applied to a Source Q column equilibrated with 10 mM Tris (pH 7.5), and the column was subjected to isocratic solvent washing using 20 mM Tris (pH 7.5) buffer solution containing 50 mM sodium chloride (NaCl) to remove impurities.
  • the IFNa-PEG-Fc protein complex was eluted with a concentration gradient of a buffer solution containing 150 mM sodium chloride (NaCl). A small amount of unreacted Fc and interferon-alpha dimer were present as impurities in the obtained IFNa-PEG-Fc protein complex fraction.
  • Source iso GE Healthcare
  • Source iso GE Healthcare was equilibrated with a 20 mM potassium phosphate (pH 6.0) buffer solution containing about 1.3 M ammonium sulfate, and then the purified IFNa-PEG-Fc protein complex fraction was applied thereto.
  • a high-purity IFNa-PEG-Fc protein complex was purified with a linear concentration gradient of a 20 mM potassium phosphate (pH 6.0) buffer solution. N-terminal selectivity of the Fc region of the prepared IFNa-PEG-Fc protein complex was examined by peptide mapping, and the selectivity was found to be 90% or higher.
  • the 17 ’ 65S G-CSF-PEG-Fc protein complex was prepared using a derivative ( 17,65 G-CSF) prepared by substituting serine for the amino acids at positions 17 and 65 of the native G-CSF, and then purified.
  • ALD-PEG-ALD (IDB, Korea), which is polyethylene glycol (PEG) having a molecular weight of 3.4 kDa and aldehyde reactive groups at both ends thereof, was added to 5 mg/mL of 17,65S G-CSF (molecular weight: 18 kDa) dissolved in 100 mM phosphate buffer at a molar ratio of G-CSF :PEG of 1 :5 to 1 : 10.
  • a reducing agent sodium cyanoborohydride (NaCNBHi, Sigma), was added thereto at a final concentration of 20 mM, and allowed to react at 4°C to 8°C under slow stirring for about 1 hour.
  • reaction mixture was subjected to SP HP (GE Healthcare) cation exchange chromatography to purify a l765S G-CSF-PEG conjugate with a high purity.
  • SP HP GE Healthcare
  • the immunoglobulin Fc fragment was added and reacted at a molar ratio of 17,65S-G-CSF-PEG conjugate: immunoglobulin Fc of 1:1 to 1:4.
  • the reaction solution was prepared as a 100 mM phosphate buffer (pH 5.5 to 6.5), and sodium cyanoborohydride (NaCNBH3, Sigma) was added as a reducing agent at a final concentration of 20 mM. The reaction was allowed at 4°C to 8°C under slow stirring.
  • the reaction mixture was buffer-exchanged to 10 mM Tris (pH 8.0) containing 2 M NaCl and then passed through a Source Phenyl column.
  • the 17,65S G-CSF-PEG-Fc protein complex was purified with a concentration gradient of 20 mM Tris (pH 8.0) buffer solution.
  • a small amount of unreacted immunoglobulin Fc and 17>65S G-CSF dimer as impurities were present in the obtained 17,65S G-CSF-PEG-FC protein complex fraction.
  • Q HP GE Healthcare anion chromatography was further performed.
  • Q HP GE Healthcare
  • a high-purity 17 ⁇ 55S G-CSF-PEG-Fc protein complex was purified with a linear concentration gradient of a 20 mM Tris (pH 8.0) buffer solution containing 1 M sodium chloride.
  • N-terminal selectivity of the Fc region of the prepared 17 65S G- CSF-PEG-Fc protein complex was examined by peptide mapping, and the selectivity was found to be 90% or higher.
  • Example 3 Preparation of protein complex using PEG with different reactive groups.
  • SMB-PEG-SMB (Nektar, USA), which is polyethylene glycol (PEG) having a molecular weight of 3.4 EDa and succinimidyl alpha-methyl butanoate (SMB) reactive groups at both ends thereof, was added to 10 mg/mL of 17,65S G-CSF (molecular weight 18 kDa) dissolved in 20 mM phosphate buffer (pH 8.0) at a molar ratio of G-CSF:PEG of 1:3, and allowed to react at room temperature under slow stifling for about 30 minutes.
  • PEG polyethylene glycol
  • SMB succinimidyl alpha-methyl butanoate
  • the immunoglobulin Fc fragment was added and reacted at a molar ratio of 17,65S G-CSF-PEG conjugate: immunoglobulin Fc of 1 :4 to 1 :8.
  • the reaction was allowed in 20 mM phosphate buffer (pH 5.5 to 6.5) at room temperature for about 2 hours under slow stifling.
  • the reaction solution was applied to a Q HP column equilibrated with 20 mM Tris (pH 8.0) buffer, and the l 765S G-CSF-PEG-Fc protein complex was purified with a concentration gradient of a buffer solution containing 1 M sodium chloride (NaCl). A small amount of unreacted immunoglobulin Fc and 17,65S G-CSF dimer as impurities was present in the obtained 17,65S G-CSF- PEG-Fc protein complex fraction. In order to remove the impurities, Source iso (GE Healthcare) hydrophobic chromatography was further performed.
  • a high-purity 17,65S G-CSF-PEG-Fc protein complex was purified with a linear concentration gradient of 50 mM Tris (pH 7.5) buffer solution containing 1.2 M ammonium sulfate using Source iso (GE Healthcare). N-terminal selectivity of the Fc region of the prepared 17,65S G-CSF -PEG-Fc protein complex was examined by peptide mapping, and the selectivity was found to be 90% or higher.
  • a FacVII-ATKAVC-PEG-Fc complex was prepared using F c VII- ATKA VC, which is a FacVII derivative of Korean Patent Application No. 10-2012-0111537 previously submitted by the present inventors.
  • the immunoglobulin Fc region and maleimide- 10 kDa PEG-aldehyde were mixed at a molar ratio of 1 : 1 in a 100 mM phosphate buffer solution (pH 5.5 to 6.5), and a reducing agent, 20 mM sodium cyanoborohydride (NaCNBI I-, Sigma), was added thereto under a protein concentration of 10 mg/mL.
  • the reaction was allowed at a low temperature (4°C to 8°C) for about 2 hours.
  • FacVII-ATKAVC-PEG-Fc complex FacVII-ATKAVC was reacted in 10 mM glycyl glycine buffer at pH 5.5 at room temperature for about 2 hours by adding 0.5 mM to 2 mM triphenylphosphine-3,3',3”-trisulfonic trisodium salt hydrate as a reducing agent so as to reduce the C-terminus.
  • the C-terminus-reduced FacVII-ATKAVC and monoPEGylated immunoglobulin Fc fragment (maleimide-10 kDa PEG- Fc) were mixed at a molar ratio of 1 :4 to 1:20, and the reaction was allowed at a total protein concentration of 1 mg/mL to 2 mg/mL in 50 mM Tris buffer at pH 7.5 at room temperature for about 2 hours.
  • Example 8-2 The reaction solution of Example 8-2 was subjected to Source Q anion chromatography, and the FacVII-ATKAVC- 10 kDa PEG-Fc complex was eluted with a concentration gradient of sodium chloride in a 20 mM Tris buffer solution at pH 7.5.
  • To activate FacVII of the FacVII- ATKAVC-PEG-Fc complex reaction was allowed in a 0.1 M Tris-HCl buffer solution at pH 8.0 under conditions of about 4 mg/mL of FacVII for about 18 hours at a low temperature (4°C to 8°C).
  • FacVIIa-ATKAVC-PEG-Fc was purified by size exclusion chromatography (GE Healthcare) using Superdex 200 in a 10 mM glycylglycine buffer solution at pH 5.5. N-terminal selectivity of the Fc region of the prepared FacVIIa-ATKAVC-PEG-Fc protein complex was examined by peptide mapping, and the selectivity was found to be 90% or higher.
  • Example 5 Preparation of protein complex using PEG with a different molecular weight
  • ALD-PEG-ALD (Nektar, USA), which is polyethylene glycol having a molecular weight of 10 kDa and reactive aldehyde groups at both ends thereof, was used to prepare and purify an insulin-10 kDa PEG conjugate in the same manner as in Example 5-2.
  • the purified insulin-10 kDa PEG conjugate was concentrated to a concentration of about 5 mg/mL and then used to prepare and purify an insulin-10 kDa PEG-Fc protein complex in the same manner as in Example 2-3.
  • the protein complexes prepared in the above Examples were analyzed by non-reduced SDS-PAGE using a 4% to 20% gradient gel and a 12% gel. SDS-PAGE analysis and Western blot analysis of individual protein complexes using antibodies against immunoglobulin Fc and physiologically active polypeptides were performed. As shown in FIG. 1, a coupling reaction resulted in the successful production of IFNa-PEG-Fc (A), hGH-PEG-Fc (B), 17 ’ 65S G-CSF-PEG- Fc (C), Insulin-PEG-Fc (D), EPO-PEG-Fc (E), CA-Exendin4-PEG-Fc (F), and FacVII-PEG-Fc (G).
  • a normocythemic mice assay was performed to measure reticulocyte levels after subcutaneous injection of EPO-PEG-Fc into normocythemic mice.
  • EPO-PEG-Fc positional isomers As shown in Table 3, comparison of in vivo activities between EPO-PEG-Fc positional isomers showed that the EPO-PEG-Fc complex of the present invention, which was prepared by specific binding to N-terminus of immunoglobulin Fc fragment, has about 40% increased titer, compared to an EPO-PEG-Fc complex which was prepared by binding to another position of an immunoglobulin Fc region.
  • the protein complex comprising the physiologically active polypeptide, the non-peptidyl polymer, and the immunoglobulin Fc region is prepared by using a specific site of the immunoglobulin Fc fragment as a binding site, the protein complex shows an improved in vivo activity of the physiologically active polypeptide.
  • Example 8 Randomized Human Trial 1765S G-CSF-PEG-Fc protein complex (Eflapegrastim ) vs. Pegfilgrastim in the Management of Chemotherapy-Induced Neutropenia in Breast Cancer Patients Receiving Docetaxel and Cyclophosphamide (TC).
  • Eflapegrastim (13.2 mg/0.6 mL; 3.6 mg GCSF equivalent) in patients with breast cancer who were candidates for adjuvant or neoadjuvant chemotherapy with docetaxel and cyclophosphamide (TC)
  • TC docetaxel and cyclophosphamide
  • Eligible patients were randomized 1: 1 to the following two treatment arms: (a) Eflapegrastim arm: Eflapegrastim 13.2 mg/0.6 mL (3.6 mg G-CSF equivalent) fixed dose and (b) Pegfilgrastim arm: Pegfilgrastim 6 mg/0.6 mL (equivalent to 6.0 mg G-CSF) fixed dose.
  • TC was administered on Day 1 of each cycle intravenously (IV) was: (i) Docetaxel at 75 mg/m 2 IV infusion per institute’s standard of care (ii) Cyclophosphamide 600 mg/m 2 IV infusion per institute’s standard of care.
  • Each treatment cycle was 21 days with up to a maximum of 4 cycles of chemotherapy.
  • Eflapegrastim or Pegfilgrastim were administered on Day 2 of each cycle, approximately 24 hours ( ⁇ 2 hours) after TC chemotherapy. Pegfilgrastim was to be administered according to the manufacturer’ s Prescribing Information (6 mg subcutaneously once per chemotherapy cycle).
  • EOT visits were performed 35( ⁇ 5) days from the last dose of study treatment.
  • CBC samples were drawn on Day 1 prior to the chemotherapy and then daily from Days 4 to 15 or until recovery from neutropenia.
  • CBC samples were drawn on Day 1 predose and then on Days 4, 7, 10, and 15 ( ⁇ 1 day for each collection). CBC was also collected at the End-of-Treatment Visit.
  • the long-term safety includes adverse event (AE) assessment via telephone at 3 months and 9 months and clinic visits for AE assessment and immunogenicity blood draw at 6 months and 12 months.
  • AE adverse event
  • the DSN in Cycle 1 is defined as the number of postdose days of severe neutropenia
  • Efficacy analysis was measured based on the Duration of Severe neutropenia (DSN) in Cycle 1 defined as the number of postdose days of severe neutropenia (ANC ⁇ 0.5> ⁇ 10 9 /L) from the first occurrence of an ANC below the threshold.
  • DSN Duration of Severe neutropenia
  • ANC severe neutropenia
  • the results showed that the mean DSN for the Eflapegrastim arm was 0.20 ( ⁇ 0.50) days compared with a mean DSN of 0.35 ( ⁇ 0.68) days in the Pegfilgrastim arm.
  • the difference in mean DSN between the Eflapegrastim arm and the Pegfilgrastim arm was -0.15 days, and the corresponding 95% CI was (-0.264, -0.032) using the percentile method as specified in the statistical analysis plan.
  • the Eflapegrastim arm to the Pegfilgrastim arm was demonstrated to provide better or as effective as Pegfilgrastim (upper bound of 95% CI ⁇ 0.62 days; / 'O.OOO l ).
  • the results demonstrated a statistical superiority of Eflapegrastim over Pegfilgrastim in cycle 1 (upper bound of 95% CI ⁇ 0; 0.038), indicating that the incidence of severe neutropenia is significantly lower in the Eflapegrastim arm (FIG. 2 and FIG. 3).
  • the incidences of adverse events were substantially were comparable between treatment groups, most of which were considered relating to the chemotherapy (TC) administration.
  • Example 9 Open-label Human Trial to Evaluate Duration of Severe Neutropenia After the Same-Pay, Varying Dosing Time Schedules of 17 ’ 65S G-CSF-PEG-Fc protein complex (Eflapegrastim) Administration in Patients with Breast-Cancer Receiving Docetaxel and Cyclophosphamide
  • Eflapegrastim on the same day as chemotherapy and to identify the optimal timing for same-day dosing
  • an open-label human trial is designed to assess the impact of different doses and dosing times on the duration of neutropenia and on absolute neutrophil counts in chemotherapy-induced neutropenic patients with breast cancer who underwent a treatment course with docetaxel and cyclophosphamide (TC).
  • Current practice is for the patient to return to the clinic approximately 24 hours after TC treatment for subcutaneous injection of a G-CSF product.
  • at least one shortcoming associated with such an approach is follow up patient compliance as they may, for example, miss their visits due to adverse events or other complications caused by the TC treatment.
  • the present trial is designed to investigate the use of Eflapegrastim when administered the same day as TC at 3 dose time schedules (30 minutes, 3 hours, and 5 hours) after TC administration.
  • Eligible patients will enter an open-label trial to the following three treatment arms: (a) arm 1 : at least 15 patients receive Eflapegrastim 13.2 mg/0.6 mL (3.6 mg G-CSF equivalent) administration is 30 minutes + 5 minutes, from the end of TC administration (b) Arm 2: at least 15 patients receive Eflapegrastim 13.2 mg/0.6 mL (3.6 mg G-CSF equivalent) administration is 3 hours + 15 minutes from the end of TC administration (c) Arm 3: at least 15 patients receive Eflapegrastim 13.2 mg/0.6 mL (3.6 mg G-CSF equivalent) administration is 5 hours + 15 minutes from the end of TC administration.
  • the TC treatment consisted of 3 cycles wherein on Day 1 of each cycle: (i) Docetaxel was administered at 75 mg/m 2 IV infusion per institute’s standard of care (ii) Cyclophosphamide 600 mg/m 2 IV infusion per institute’s standard of care. Each treatment cycle was 21 days, with up to 3 cycles of chemotherapy.
  • eligible patients must have a new diagnosis of histologically confirmed early-stage breast cancer (ESBC), defined as operable Stage I to Stage IIIA breast cancer and a candidate to receive adjuvant or neoadjuvant TC chemotherapy. Further, they must have an adequate hematological, renal, and hepatic function as defined by (a) ANC >1.5> ⁇ 10 9 /L, (b) Platelet count >100xl0 9 /L, (c) Hemoglobin >10 g/ dL, (d) Calculated creatinine clearance >50 mL/min, and (e) Total bilirubin ⁇ 1.5 mg/dL and (f) )AST)/serum glutamic-oxaloacetic transaminase (SGOT) and alanine aminotransferase (ALT)Zserum glutamic-pyruvic transaminase (SGPT) ⁇ 2.5*ULN, and alkaline phosphatase ⁇ 2.0/UL
  • Eflapegrastim The dose of Eflapegrastim to be administered is a fixed-dose 13.2 mg/0.6 mL containing 3.6 mg G-CSF per cycle. However, in Cycle 1, Eflapegrastim is administered on the same day as chemotherapy administration and 24 ( ⁇ 3) hours from the end of TC administration in Cycles 2 to 4.
  • a complete physical examination including a description of external signs of the neoplastic disease and co-morbidities, is performed at the Screening Visit and at the End-of- Treatment Visit. Symptom-directed physical examinations are conducted at all other visits. Physical examinations are to be completed by a physician or their designee qualified to perform such examinations.
  • NCI National Cancer Institute
  • CCAE Common Terminology Criteria for Adverse Events
  • patients receiving Eflapegrastim at 30 minutes + 5 minutes, 3 hours + 15 minutes, and 5 hours + 15 minutes from the end of TC administration, at 0.6 mL (3.6 mg G-CSF equivalent) doses exhibit the same or shorter duration of neutropenia as compared to those that receive Eflapegrastim after 24 hours after the end of the TC administration.
  • the Examples provided herein support the superiority of the G-CSF protein complex with the immunoglobulin Fc region attached through a PEG moiety to increase in vivo duration of the physiologically active polypeptide and to increase or maintain in vivo activity (potency) at the same time.
  • Example 10 Eflapegrastim Enhanced Efficacy Compared to Pegfilgrastim in Neutropenic Rats Supports Potential for Same-Pay Dosing
  • neutropenia Major dose-limiting toxicity of chemotherapy in 43% of patients not given myeloid growth factors is neutropenia, with 24% of these patients having severe neutropenia. In addition, severe febrile neutropenia in 9% of patients not treated with growth factors after chemotherapy. Febrile neutropenia increases the risk of infection, leading to patient hospitalization, morbidity, and mortality and can also lead to chemotherapy dose reductions and drug holidays that can result in significantly reduced chemotherapy efficacy.
  • G-CSF is known to stimulate the proliferation of bone marrow progenitor cells and enhance neutrophil production in vitro and in vivo.
  • the administration of exogenous G-CSF to patients receiving myelosuppressive chemotherapy increases neutrophil counts and results in resolution of chemotherapy-induced neutropenia.
  • clinical practice guidelines recommend prophylactic administration of G-CSF 24 hours after the end of chemotherapy to reduce the degree of neutropenia and FN.
  • Pegfilgrastim was the first US FDA-approved long-acting G-CSF, developed by pegylating filgrastim at the N-terminal methionine residue with a 20 kDa polyethylene glycol (PEG) molecule.
  • PEG polyethylene glycol
  • ASCO American Society of Clinical Oncology
  • Eflapegrastim (HM10460A, SPI-2012, Rolontis®; Spectrum Pharmaceuticals, Irvine, CA, USA and Hanmi Pharmaceuticals, Seoul, South Korea) is a novel, long-acting recombinant human (rh) G-CSF analog currently in late-stage clinical development. Eflapegrastim differs from the currently approved long-acting G-CSF, Pegfilgrastim, due to the conjugation of G-CSF to human IgG4 Fc fragment.
  • the Fc fragment is expressed as a homodimer, and the conjugate has a molecular weight of approximately 72 kD, since only one of the two polypeptide chains of the Fc fragment is conjugated to a single molecule of G-CSF analog (USAN adoption statement).
  • the IgG4 Fc fragment imparts neonatal Fc receptor (FcRn)-mediated protection from degradation, increased uptake of Eflapegrastim into bone marrow, and improved efficacy.
  • Eflapegrastim was provided by Hanmi Pharmaceuticals. Pegfilgrastim was purchased from Amgen, Inc, Thousand Oaks, CA, USA. The doses and concentrations of Eflapegrastim and Pegfilgrastim were expressed as a standardized dose of G-CSF.
  • Human serum immunoglobulin G (IgG, I. V. Globulin S) was purchased from Green Cross Corporation, Korea.
  • RPMH640 (cat. No.: 22400), IMDM;(cat. No.: 12440), FBS (cat. No.: 10082), penicillinstreptomycin (cat. No.: 15140), HEPES buffer (Cat. No. 11344-041), and trypsin-EDTA were purchased from Gibco.
  • EMEM (cat. No.: 30-2003) was from ATCC Media Products, and G418 Sulphate (cat. No. 61-234-RG) was obtained from Cellgro.
  • Sodium pyruvate (cat. No.: P4562) and glutamine (cat. No.: G5792) were procured from Sigma.
  • D-phosphate buffered saline (PBS) (cat. No.: LB 001-02) was purchased from Welgene. 10.3 G-CSF receptor binding
  • CM5 biosensor chip (BR-1006-68, GE Healthcare).
  • the CM5 sensor chip surface was activated by injecting a 1: 1 mixture of 0.1 mol/L N-hydroxysuccinimide (GE Healthcare) and 0.1 mol/L 3-(N,N-dimethylamino)propyl-N- ethyl carbodi ami de (GE Healthcare).
  • Eflapegrastim 5.5 - 88 nmol/L
  • Pegfilgrastim 6.25 - 100 nmol/L
  • HBS-P buffer 10 mmol/L HEPES, pH 7.4; 150 mmol/L NaCl; 0.005% polysorbate 20
  • Eflapegrastim and Pegfilgrastim were determined by measuring [methyl- 3 H] thymidine (Amersham, TRA-120-lMCi) incorporation in mouse bone marrow cells obtained from femurs of 4-6 weeks old C57BL/6NCrl mice (Korea Orient Bio Inc., Charles River agency).
  • Non-adherent bone marrow cells were incubated with serial 3 -fold dilutions of Eflapegrastim or Pegfilgrastim in RPML1640 media at 37 °C, 5% CO2 and 95% RH for 54 hours; [methyl- 3 H] thymidine (25 pL, 0.25 pCi/well) was added, and incubation continued for an additional 18 hours.
  • the cells were harvested onto a Unifilter-96 GF/C plate with a filter mat using a Uniflter-96 harvester (PerkinElmer) and washed.
  • FcyRI binding assay FcyRI was coated on 96-well microplate, serial dilutions of test materials in assay buffer (phosphate-buffered saline, pH 7.4) were added to the wells and incubated for 90 - 120 min. at ⁇ 25°C (RT). The plates were then washed with assay buffer containing 0.05% Tween 20 and incubated with HRP-conjugated goat anti-human IgG for 90 min. After washing, color was developed using 3,3 ’,5, 5 ’-tetramethylbenzidine (TMB; BD bioscience) substrate, and the absorbance was measured at 450 nm using a microplate reader.
  • assay buffer phosphate-buffered saline, pH 7.4
  • MDCK Madin-Darby canine kidney
  • Eflapegrastim or Pegfilgrastim diluted to 10 pM with the assay medium was loaded into the upper wells and were incubated for 1 hour at 37 °C in 95% 02/5% CO2.
  • the quantity of Eflapegrastim and Pegfilgrastim transported through the cell layer to the lower wells was determined by ELISA using a human G-CSF ELISA kit (IBL, 27131).
  • chemotherapy-induced neutropenia was induced using docetaxel (4 mg/kg) and CPA (32 mg/kg) (TC) administered i.p. followed by the administration of G-CSF s.c. concomitant to chemotherapy and at 2, 5, and 24 hours post chemotherapy.
  • venous blood samples were collected for determination of absolute neutrophil count (ANC) (Sysmex XN1000-V (Sysmex, Japan) as outlined in Table 4 for time to recovery.
  • ANC absolute neutrophil count
  • AUECANC ANC-versus-time effect curve above baseline
  • Chemotherapy-induced neutropenia was induced in male SD rats by administering CPA (50 mg/kg, i.p.). Four days later, Eflapegrastim (100 pg/kg as G-CSF) or Pegfilgrastim (100 pg/kg as G-CSF) was administered s.c. to 15 animals per group. At 8, 30, 54, 72, and 96 hours post G- CSF therapy, three animals per group were euthanized, and serum and bone marrow G-CSF levels were determined via ELISA (IBL Cat. No. JP27131).
  • FcyRs Fey receptors
  • glycan moieties in the Fc fragment are known to interfere with FCyR binding and elicit immune effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • Fc fragment of Eflapegrastim manufactured from recombinant A. coli cells, is aglycosylated and not expected to bind to FcyRs or Clq. Results confirmed that Fc fragment of Eflapegrastim and Eflapegrastim failed to bind to various FcyRs (FIGS. 4A, 4B, and 4C) or FcyRs expressed in U937 cells (FIG. 4D) or Clq (FIG. 4E). 10.14 Eflapegrastim binds to FcRn and undergoes FcRn-mediated transcytosis
  • the FcRn binds to the Fc domain of IgG and facilitates its transport across endothelial and epithelial barriers. 17 18 Hence it was of interest to determine if Eflapegrastim binds to FcRn. Using an ELISA method, Eflapegrastim was shown to bind to FcRn with affinities comparable to those of glycosylated and aglycosylated Fc fragments of human IgGl, at levels which are lower than that of serum IgG (FIGS. 5A and 5B).
  • FIGS. 5A and 5B show a 4-fold increase in transport of Eflapegrastim in FcRn-expressing MDCK cells compared to MDCK-WT cells (43 versus 160 pM), while the transport of Pegfilgrastim was similar in FcRn-expressing MDCK and MDCK-WT cells (73 versus 86 pM).
  • Eflapegrastim and Pegfilgrastim were evaluated in chemotherapy-induced neutropenic rats in two studies.
  • the efficacy of Eflapegrastim and Pegfilgrastim administered 24 hours after chemotherapy was first evaluated (FIGS. 6A, 6B, and 6C) in accordance with ASCO guidelines.
  • Eflapegrastim doses, expressed as G-CSF equivalent, ranging from 9 to 88 pg/kg were compared with a Pegfilgrastim dose of 100 pg/kg (as G-CSF), the Pegfilgrastim dose that was found to be effective in stimulating bone marrow cell proliferation in rats.
  • FIG. 6A The ANC versus time profiles are shown in FIG. 6A.
  • administration of CPA resulted in neutropenia (ANC values below the mean ANC of the untreated control group) with a duration of 6-8 days (FIG. 6).
  • Treatment with Pegfilgrastim (100 ug/kg as G-CSF) and Eflapegrastim (9 to 88 pg/kg as G-CSF) 24 hours after CPA (FIG. 6A) produced an initial increase in ANC above baseline during the first 12 to 24 hours after injection, which then rapidly declined reaching a nadir on Day 2 or 3, increased again reaching a peak on Day 4 or 5 and declined by Day 6 or 7.
  • the Pegfilgrastim treated group as well as different dose groups of Eflapegrastim showed statistically significant increases in AUECANC (FIG. 6B) and decreases in DN (FIG. 6C) compared to vehicle control.
  • Treatment with Eflapegrastim resulted in a dose-dependent increase in AUECANC (FIG. 6B) and a decrease in DN (FIG. 6C) as the dose was increased from 9 pg/kg to 53 pg/kg, but there was no further significant increase in AUECANC or decrease in DN as the dose was increased to 88 pg/kg (FIGS. 6B and 6C).
  • FIGS. 7A-D show the ANC profiles following administration of Eflapegrastim or Pegfilgrastim to rats at 0, 2, 5, and 24 hours after chemotherapy, respectively.
  • administration of docetaxel-cyclophosphamide resulted in neutropenia with a duration of 6-8 days.
  • Treatment with Pegfilgrastim or Eflapegrastim elicited an initial increase in ANC above baseline during the first 12 to 24 hours post-treatment, then rapidly declined reaching a nadir on Day 3.
  • post-nadir ANC reached a peak on Day 5 or 6 and declined thereafter.
  • Eflapegrastim treated rats showed significantly higher AUECANC and lower DN compared to Pegfilgrastim treated rats, regardless of time of administration after chemotherapy (FIGS. 7E and 7F). 10.16 Eflapegrastim reaches higher levels in serum and bone marrow in chemotherapeutic- induced neutropenic rats
  • Eflapegrastim In view of the superior efficacy of Eflapegrastim compared with Pegfilgrastim in a neutropenic rat model, the distribution of Eflapegrastim into the bone marrow of neutropenic rats was compared with Pegfilgrastim. Concentrations of Eflapegrastim and Pegfilgrastim in serum and in bone marrow were determined at different times following s.c. administration of the test article (Table 5). Eflapegrastim and Pegfilgrastim reached peak concentrations in serum and bone marrow at 30 hours after s.c. administration.
  • Eflapegrastim exhibited approximately 3-fold higher exposure (AUCiast) and peak concentration (Cmax) than Pegfilgrastim at similar doses (AUCiast 12401vs 4263 ng hr/ml; Cmax 308 versus 125 ng/ml, respectively).
  • the terminal half-lives for both compounds were comparable at approximately 4 hours.
  • the absolute concentrations of Eflapegrastim in bone marrow were higher than those of Pegfilgrastim at all corresponding time points, and the differences were statistically significant in bone marrow at 30 and 54 hours. Eflapegrastim concentrations also declined at a slower rate than Pegfilgrastim in the bone marrow.
  • Eflapegrastim is a novel long-acting G-CSF with unique structural features compared to currently approved long-acting pegylated G-CSF products.
  • Eflapegrastim contains an aglycosylated IgG4-Fc fragment conjugated to a human recombinant G-CSF analog via a short PEG linker.
  • the strategy behind this structural modification to G-CSF is to increase the half-life and the ability to penetrate to the site of action without adversely altering affinity or potency. Binding studies with G-CSF receptors demonstrated that Eflapegrastim and Pegfilgrastim have comparable affinities.
  • Chemotherapy may cause myelosuppression, potentially reducing bone marrow stem cell proliferation and subsequently decreased ANC.
  • G-CSF treatment improves bone marrow proliferation, myeloid progenitor cell activation, and neutrophil differentiation and migration.
  • Eflapegrastim and Pegfilgrastim displayed similar binding affinity in vitro, suggesting that the addition of the Fc fragment or PEG linker did not perturb G-CSF binding.
  • the Fc fusion protein in Eflapegrastim has the potential to trigger ADCC by interaction of the Fc fragment with Fey receptors on NK cells or neutrophils, no binding of Eflapegrastim to Fey receptors was observed (FIG. 3), indicating that Eflapegrastim does not exert ADCC.
  • the neonatal Fc receptor for IgG (FcRn) binds to the Fc portion of IgG and contributes to effective IgG recycling and transcytosis, thereby enhancing tissue residence.
  • FcRn is highly expressed on bone marrow-derived cells and myeloid-derived antigen-presenting cells. Eflapegrastim showed strong binding to FcRn (FIG. 5A) and FcRn dependent transcytosis (FIG. 5B) suggesting, enhanced uptake and retention of Eflapegrastim as compared to Pegfilgrastim in the bone marrow. (Table 5).
  • Eflapegrastim showed strong FcRn binding ability resulting in increased uptake and longer duration of residence in the bone marrow, compared to Pegfilgrastim. It was therefore hypothesized that same-day dosing of Eflapegrastim might overcome the loss of Pegfilgrastim effectiveness.
  • two studies were performed in chemotherapy-induced neutropenic rats. In the first study, Eflapegrastim and Pegfilgrastim were administered 24-hours post-chemotherapy with CPA as per ASCO guidelines. Eflapegrastim elicited a dose-dependent blunting of the chemotherapy-induced neutropenia with a reduction in the neutrophil nadir and an increased rate of neutrophil recovery (FIG. 6).
  • Pegfilgrastim was also effective in this dose regimen; however, the magnitude of the response was less than that of Eflapegrastim. It was observed that when Eflapegrastim was administered concomitantly with chemotherapy or up to 5 hours post-chemotherapy, there was a more profound reduction in the degree of neutropenia and a more rapid rate of recovery of ANC compared to Pegfilgrastim. When either Eflapegrastim or Pegfilgrastim was administered the day after chemotherapy with CPA and docetaxel (FIG. 7), similar effects were observed as with CPA alone (FIG. 6).
  • Eflapegrastim may have enhanced its bioavailability when the myeloid progenitors were regenerated post-chemotherapy. Therefore, there is the potential for Eflapegrastim to be effective when dosed to patients the same day as chemotherapy.
  • Eflapegrastim and Pegfilgrastim have similar in vitro binding affinity.
  • the FcRn fragment in Eflapegrastim increases the uptake of the drug into bone marrow resulting in increased potency in chemotherapy -induced neutropenia.
  • the greater bone marrow exposure and retention to Eflapegrastim resulted in a decrease in the duration of neutropenia when compared to Pegfilgrastim.
  • CPA cyclophosphamide
  • DPBS Dulbecco’s phosphate-buffered saline
  • G-CSF Granulocytecolony stimulating factor
  • i.p intraperitoneal
  • s.c subcutaneous.

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Abstract

La présente invention concerne une méthode pour prévenir, soulager ou traiter une affection (c'est-à-dire une neutropénie) chez un sujet qui en a besoin, l'affection étant caractérisée par une altération de la production des globules blancs chez le sujet. La méthode consiste à administrer au sujet une quantité thérapeutiquement efficace d'un complexe protéique le même jour qu'un protocole de chimiothérapie, le complexe protéique étant un facteur de stimulation des colonies de granulocytes humains (hG-CSF) modifié lié de manière covalente à une région Fc d'immunoglobuline par l'intermédiaire d'un polymère non peptidylique. Le polymère non peptidylique est lié spécifiquement à un site à une extrémité N-terminale de la région Fc d'immunoglobuline, et le hG-CSF modifié comprend des substitutions au niveau d'au moins l'un de Cys17 et de Pro65.
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US11684655B2 (en) 2019-05-31 2023-06-27 Spectrum Pharmaceuticals, Inc. Methods of treating neutorpenia using G-CSF protein complex

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WO2002028896A1 (fr) * 2000-09-18 2002-04-11 Isis Innovation Limited Analogues du facteur de stimulation de colonies de granulocytes humains (g-csf)
US20180326013A1 (en) * 2015-09-24 2018-11-15 Hanmi Pharm. Co., Ltd Protein complex by use of a specific site of an immunoglobulin fragment for linkage
US20200038395A1 (en) * 2017-02-01 2020-02-06 Beyondspring Pharmaceuticals, Inc. Method of reducing neutropenia

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WO2002028896A1 (fr) * 2000-09-18 2002-04-11 Isis Innovation Limited Analogues du facteur de stimulation de colonies de granulocytes humains (g-csf)
US20180326013A1 (en) * 2015-09-24 2018-11-15 Hanmi Pharm. Co., Ltd Protein complex by use of a specific site of an immunoglobulin fragment for linkage
US20200038395A1 (en) * 2017-02-01 2020-02-06 Beyondspring Pharmaceuticals, Inc. Method of reducing neutropenia

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MASAHARU ISHIKAWA, HIROSHI IIJIMA, RIKA SATAKE-ISHIKAWA, HARUHIKO TSUMURA, AKIHIRO IWAMATSU, TOSHIHIKO KADOYA, YOSHIHIRO SHIMADA, : "The Substitution of Cysteine 17 of Recombinant Human G-CSF with Alanine Greatly Enhanced its Stability", CELL STRUCTURE AND FUNCTION, vol. 17, no. 1, 1 January 1992 (1992-01-01), pages 61 - 65, XP002636941, ISSN: 0386-7196, DOI: 10.1247/csf.17.61 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11684655B2 (en) 2019-05-31 2023-06-27 Spectrum Pharmaceuticals, Inc. Methods of treating neutorpenia using G-CSF protein complex

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