WO2017095201A1 - 지방산 유도체를 이용한 단백질 결합체 및 이의 제조방법 - Google Patents
지방산 유도체를 이용한 단백질 결합체 및 이의 제조방법 Download PDFInfo
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- WO2017095201A1 WO2017095201A1 PCT/KR2016/014144 KR2016014144W WO2017095201A1 WO 2017095201 A1 WO2017095201 A1 WO 2017095201A1 KR 2016014144 W KR2016014144 W KR 2016014144W WO 2017095201 A1 WO2017095201 A1 WO 2017095201A1
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- 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
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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
- C07K14/575—Hormones
- C07K14/605—Glucagons
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/2264—Obesity-gene products, e.g. leptin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/2278—Vasoactive intestinal peptide [VIP]; Related peptides (e.g. Exendin)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/26—Glucagons
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- 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
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- 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
- A61K47/54—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 compound
- A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- 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
- A61K47/62—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 a protein, peptide or polyamino acid
- A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
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- 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
- A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
- A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
- A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
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- 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
- A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- 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
Definitions
- the present invention relates to a protein conjugate in which a physiologically active polypeptide and a biocompatible substance are linked through fatty acid derivatives, and whose duration of physiological activity is longer than that of the natural form, and a method for producing the same.
- physiologically active polypeptides have low stability and are readily denatured and degraded by proteolytic enzymes in the blood and are easily removed through the kidneys or liver. Therefore, it is necessary to frequently administer protein drugs to patients in order to maintain blood levels and titers of protein drugs containing physiologically active polypeptides as pharmacological components.
- protein medicines that are administered to patients in the form of mostly injections, frequent injections to maintain blood levels of active polypeptides can cause tremendous pain in the patient.
- efforts have been made to maximize the drug efficacy by increasing the blood stability of protein drugs and maintaining high blood drug concentrations for a long time.
- Such long-acting formulations of protein drugs should enhance the stability of the protein drugs while not inducing an immune response in the patient.
- PEG polyethylene glycol
- the binding of PEG may increase the stability of the protein, but the activity of the bioactive protein is significantly lowered, and as the molecular weight of PEG increases, the reactivity with the protein decreases and the yield decreases, and the increase in half-life is also sufficient. It is not known.
- the present inventors further stabilize the bioactive polypeptide by covalently binding a biocompatible substance which increases the in vivo stability of the bioactive polypeptide and the protein using a fatty acid derivative as a linker, rather than increasing protein stability through PEGylation,
- the present invention has been completed by recognizing that renal loss can be inhibited to significantly increase blood half-life of a physiologically active polypeptide.
- One object of the present invention is to provide a protein conjugate wherein the bioactive polypeptide and the biocompatible material are linked through a fatty acid derivative.
- Another object of the present invention is to provide a method for preparing the protein conjugate.
- the bioactive polypeptide and the biocompatible material may be linked through a reactor of a fatty acid that is a linker.
- the protein conjugate to which the biocompatible material, the fatty acid derivative and the bioactive polypeptide of the present invention are linked has been found to increase the blood half-life of the bioactive polypeptide, and thus can be widely used in the field of protein drugs.
- 1 is an SDS-PAGE of an immunoglobulin Fc conjugate prepared according to one embodiment of the present invention and a triple activator activating GLP-1, glucagon and GPI receptors at the same time, and an immunoglobulin Fc conjugated through a fatty acid derivative.
- M represents a marker
- 1 represents a non-reducing immunoglobulin Fc conjugate
- 2 and 3 represent a non-reducing and reducing protein conjugate, respectively.
- Figure 2 is a diagram showing the in vivo pharmacokinetic measurement results of the immunoglobulin Fc conjugate prepared in accordance with an embodiment of the present invention, and a conjugate in which the triple activator and the immunoglobulin Fc are linked through a fatty acid derivative. Specifically, the comparative group using the existing triple activator, showing the change in blood concentration of the drug over time.
- One embodiment of the invention is a protein conjugate wherein the bioactive polypeptide and the biocompatible material are linked via fatty acid derivatives.
- the protein conjugate according to the present invention is characterized in that the bioactive polypeptide and the biocompatible material are covalently linked to the fatty acid derivatives, respectively.
- the fatty acid derivative acts as a linker linking the bioactive polypeptide and the biocompatible material.
- the fatty acid derivative included in the protein conjugate according to the present invention has two or more reactors directly connected to the fatty acid backbone or through a linker, and is connected to the bioactive polypeptide and the biocompatible material through the reactor, respectively. It is done.
- the reactors of the fatty acid derivatives included in the protein conjugates according to the present invention are each independently 2,5-dioxopyrrolidinyl, 2,5-dioxopyrrolyl, aldehyde, aryldisulfide, heteroaryldisulfide, halogenated acet Amide (haloacetamide), or C 7-10 alkynyl (alkynyl) is characterized in that.
- the reactor of the fatty acid derivative included in the protein conjugate according to the present invention is maleimide, N-hydrosuccinimide, succinimide, C 1-4 alkylene aldehyde, orthopyridyl disulfide; OPSS), iodide acetamide (IA), halogenated acetamide containing bromine, fluorine, chlorine, or astaxin in place of the iodine, difluorocyclooctyne (DIFO), dibenzocyclooctyne (DIBO) ), Dibenzo-aza-cyclooctyne (DIBAC or DBCO), biarylazacyclooctynones (BARAC), tetramethylthiacycloheptyne (TMTH), bicyclononine; BCN) Sondheimer diyne, cyclooctyne (OCT), monofluorinated cyclooctyne (MO
- the linker of the fatty acid derivative included in the protein conjugate according to the present invention is characterized in that it comprises a C 1-3 alkylamino, (C 1-3 alkoxy) n (C 1-3 alkylamino) chain. .
- At least one linker of a bioactive polypeptide and a fatty acid derivative is linked to a biocompatible material included in the protein conjugate according to the present invention.
- the bioactive polypeptide included in the protein conjugate according to the present invention is a hormone, cytokine, interleukin, interleukin binding protein, enzyme, antibody, growth factor, transcription regulator, blood factor, vaccine, structural protein, ligand Protein or receptor, cell surface antigen or receptor antagonist.
- the bioactive polypeptide included in the protein conjugate according to the present invention is glucagon-like peptide-1 (GLP-1), glucagon, GIP (Gastric inhibitory polypeptide), auxintomodulin, xenin, insulin Incretins that regulate blood sugar and weight in the stomach or intestine, such as CCK (Cholecystokinin) amylin, gastrin, ghrlin, ghrelin, and peptide YY; Adipokines secreted from fat, such as leptin, adiponectin, adiponin, adipolin, apelin, cartonectin; Neuropeptides (neuropeptides) that are secreted from the brain, such as Keithpeptin and Nesfatin-1; Peptides or proteins secreted from muscles such as irisin, myonectin, decorin, follistatin, and muslin; Vasoactive intestinal peptides, na
- bioactive polypeptide according to the invention is characterized by activating two or more receptors simultaneously.
- the bioactive polypeptide according to the invention is characterized in that it is selected from derivatives of the naturally occurring bioactive polypeptide that is not present in its native form.
- Derivatives of physiologically active polypeptides have altered their binding capacity to specific receptors through chemical substitution such as amino acid substitution, insertion, deletion, sugar chain addition, sugar chain deletion, non-natural amino acid insertion, ring insertion, methyl residue, Phosphorus properties, ie increased water solubility, immunogenicity, such as a change in the nature means, including peptides of artificial engineered to bind to two or more different receptors.
- the biocompatible material included in the protein conjugate according to the present invention is polyethylene glycol (PEG), cholesterol, albumin and fragments thereof, albumin binding material, polymers of repeating units of specific amino acid sequences, antibodies, antibodies Fragments, FcRn binding material, in vivo connective tissue or derivatives thereof, nucleotides, fibronectin, transferrin (transferrin), saccharides (saccharides), characterized in that selected from the group consisting of polymers and combinations thereof.
- the FcRn binding material included in the protein conjugate according to the present invention is characterized in that the polypeptide comprises an immunoglobulin Fc region.
- the immunoglobulin Fc region included in the protein conjugate according to the present invention is characterized in that it is non-glycosylated.
- the immunoglobulin Fc region included in the protein conjugate according to the present invention is further characterized by a hinge region.
- the immunoglobulin Fc region included in the protein conjugate according to the present invention is selected from the group consisting of IgG, IgA, IgD, IgE, IgM, combinations thereof, and hybrids thereof. It is done.
- the immunoglobulin Fc region included in the protein conjugate according to the present invention is an IgG4 Fc fragment.
- the fatty acid derivative included in the protein conjugate according to the present invention is characterized in that the derivative is a saturated or unsaturated fatty acid having 1 to 40 carbon atoms.
- the fatty acid included in the protein conjugate according to the present invention is formic acid (HCOOH, form acid), acetic acid (CH 3 COOH), propionic acid (C 2 H 5 COOH), butyric acid (C 3 H 7 COOH), Valeric Acid (C 4 H 9 COOH), Caproic Acid (C 5 H 11 COOH), Enanthate (C 6 H 13 COOH), Caprylic Acid (C 7 H 15 COOH), Pelagonic Acid (C 8 H 17 COOH ), Capric acid (C 9 H 19 COOH), undecyl acid (C 10 H 21 COOH), lauric acid (C 11 H 23 COOH), tridecyl acid (C 12 H 25 COOH), myristic acid (C 13 H 27 COOH), pentadecyl acid (C 14 H 29 COOH), palmitic acid (C 15 H 31 COOH), heptadecylic acid (C 16 H 33 COOH), stearic acid (C 17 H 35 COOH), nona Decanoic acid (C 18
- the fatty acid included in the protein conjugate according to the present invention is characterized in that the fatty acid of palmitic acid, myristic acid, stearic acid or oleic acid or a derivative thereof.
- Another aspect of the present invention provides a method for producing a bioactive peptide comprising: (a) connecting a bioactive peptide and a biocompatible material through a fatty acid derivative having two or more reactors; And (b) separating the protein conjugate which is the result of the reaction of step (a).
- step (a3) by connecting the other one of the biocompatible material and the physiologically active polypeptide to another reactor of the fatty acid derivative of the linker separated in step (a2), wherein the reactor of the fatty acid derivative is biocompatible with the physiologically active polypeptide, respectively.
- step (a1) and (a3) in the production method according to the invention is characterized in that it is carried out in the presence of a reducing agent.
- the reducing agent in the production method according to the invention is characterized in that the sodium cyano borohydride (NaCNBH 3 ), sodium borohydride, dimethylamine borate, picoline borane complex or borane pyridine .
- reaction molar ratio of the physiologically active polypeptide and the fatty acid derivative in the step (a1) in the preparation method according to the present invention is 1: 1 to 1:20
- reaction molar ratio of the biocompatible material and the fatty acid derivative is 1: It is characterized by being 1 to 1:20.
- reaction molar ratio of the linker separated in step (a3) to step (a2) and the biocompatible material or bioactive polypeptide in the preparation method according to the present invention is 1: 0.5 to 1:20. It is done.
- One aspect of the invention provides a protein conjugate wherein the bioactive polypeptide and the biocompatible material are linked via a fatty acid derivative.
- the protein conjugate of the present invention may be one in which the physiologically active polypeptide and the biocompatible material are covalently linked to the fatty acid derivative, respectively.
- physiologically active polypeptide may be one component that forms a moiety of the conjugate.
- physiologically active polypeptide refers to a polypeptide having a certain physiological function in vivo and has a polypeptide structure. Have commonalities and various biological activities.
- the physiologically active polypeptides regulate genetic expression and physiological function, and include those that correct the abnormal condition due to the lack or excessive secretion of substances involved in function regulation in vivo. It may include.
- the bioactive polypeptide is a concept including all derivatives thereof in addition to the natural polypeptide.
- the bioactive polypeptide is not particularly limited as long as it is a bioactive polypeptide capable of exhibiting half-life increase in blood through the conjugate structure of the present invention.
- Bioactive polypeptides of the present invention are hormones, cytokines, interleukins, interleukin binding proteins, enzymes, antibodies, growth factors, transcriptional regulators, blood factors, vaccines, structural proteins, ligand proteins or receptors, cell surface antigens, receptor antagonists It is characterized by that.
- the bioactive polypeptide included in the protein conjugate according to the present invention is glucagon-like peptide-1 (GLP-1), glucagon, GIP, auxintomodulin, jenin, insulin, CCK amylin, gastrin, ghrelin Incretins that control blood sugar and weight in the stomach or intestine, such as, PYY; Adipocaines secreted from lipids such as leptin, adiponectin, adiporin, aperin, cartonectin; Neuropeptides secreted from the brain, such as kisspeptin and nesphatin-1; Peptides or proteins secreted from muscles such as iricin, myonectin, decorin, follistatin, and muslin; Vascular Actuated Growth Peptides, Natriuretic Peptides, Neutrophil Growth Factor (G-CSF), Human Growth Hormone (hGH), Erythropoietin (EPO),
- the bioactive polypeptide according to the invention is characterized in that it is selected from derivatives of the naturally occurring bioactive polypeptide that is not present in its native form.
- Derivatives of physiologically active polypeptides have altered their binding capacity to specific receptors through chemical substitution such as amino acid substitution, insertion, deletion, sugar chain addition, sugar chain deletion, non-natural amino acid insertion, ring insertion, methyl residue, Phosphorus properties, ie increased water solubility, immunogenicity, such as a change in the nature means, including peptides of artificial engineered to bind to two or more different receptors.
- physiologically active polypeptide As used herein, the term “physiologically active polypeptide”, “physiologically active protein”, “active protein” or “protein drug” refers to a polypeptide or protein that exhibits antagonistic action on physiological phenomena in vivo. Can be.
- the "biocompatible material” may be one component that forms a moiety of the conjugate, and means a material that can be combined with a bioactive polypeptide to increase the half-life in vivo.
- biocompatible material is a material capable of extending the half-life in vivo and may be used interchangeably as represented by “carrier”.
- the biocompatible material or carrier includes any material that can be combined with a physiologically active polypeptide to extend its half-life, for example polyethylene glycol (PEG), cholesterol, albumin and fragments thereof, albumin binding material, specific Consisting of polymers, antibodies, antibody fragments, FcRn binding substances, in vivo connective tissues or derivatives thereof, nucleotides, fibronectins, transferrins, saccharides, polymers, and combinations thereof It may be selected from the group, but is not limited thereto.
- the FcRn binding agent may be a polypeptide including an immunoglobulin Fc region, for example, IgG Fc.
- the biocompatible material or carrier may be bound to a bioactive polypeptide via a fatty acid derivative.
- polyethylene glycol When polyethylene glycol is used as a carrier, it may include Ambrx's Recode technology that can attach polyethylene glycol in a specific position, and may include Neose's glycopegylation technology that can specifically attach to a sugar chain. .
- releasable PEG technology in which polyethylene glycol is slowly removed in vivo may be included in the releasable PEG technology, but is not limited thereto and may include technologies that increase the bioavailability using PEG.
- Polymeric polymers can also be bonded to the conjugates of the present invention by the techniques described above.
- the protein conjugate of the present invention may be a conjugate in which albumin or an albumin fragment is directly covalently bonded to a fatty acid derivative, and a substance which binds to albumin even if albumin is not directly bound, for example, albumin specific.
- the binding antibody or antibody fragment may bind to an albumin by binding to a fatty acid derivative.
- it may be a protein conjugate coupled to a specific peptide / protein / compound having a binding force to the albumin and the fatty acid derivative, and may be a protein conjugate to which the fatty acid itself having a binding force to albumin is bound, but is not limited thereto.
- an antibody or an antibody fragment may be used as a carrier, which may be an antibody or an antibody fragment having an FcRn binding site, and an antibody fragment that does not include an FcRn binding site such as a Fab, but is not limited thereto.
- an immunoglobulin Fc region may be used as a carrier.
- immunoglobulin Fc regions are biodegradable polypeptides that are metabolized in vivo, they are safe for use as carriers of drugs.
- the immunoglobulin Fc region is advantageous in terms of the preparation, purification and yield of the conjugate because of its relatively low molecular weight compared to the whole immunoglobulin molecule, as well as the removal of Fab moieties that exhibit high heterogeneity because the amino acid sequence varies from antibody to antibody. It is also expected that the homogeneity of the drug is greatly increased and the likelihood of inducing blood antigens is lowered.
- immunoglobulin Fc region means a heavy chain constant region, excluding heavy and light chain variable regions, heavy chain constant region 1 (CH1) and light chain constant region (CL1) of an immunoglobulin.
- the Fc fragment may also include a hinge portion in the heavy chain constant region.
- the immunoglobulin Fc region of the present invention may be an extended Fc fragment including some or all heavy chain constant region 1 (CH1) and / or light chain constant region 1 (CL1), except for the heavy and light chain variable regions of the immunoglobulin. Can be.
- CH1 heavy chain constant region 1
- CL1 light chain constant region 1
- immunoglobulin Fc regions are biodegradable polypeptides that are metabolized in vivo, they are safe for use as carriers of drugs.
- the immunoglobulin Fc region is advantageous in terms of the preparation, purification and yield of the conjugate because of its relatively low molecular weight compared to the whole immunoglobulin molecule, and also removes the Fab moiety that exhibits high heterogeneity because the amino acid sequence varies from antibody to antibody. It is also expected that the homogeneity will be greatly increased and the likelihood of inducing blood antigens will be lowered.
- the immunoglobulin Fc region includes not only a native amino acid sequence but also sequence variants thereof.
- Amino acid sequence variants mean that one or more amino acid residues in a natural amino acid sequence have different sequences by deletion, insertion, non-conservative or conservative substitution, or a combination thereof.
- IgG Fc amino acid residues 214 to 238, 297 to 299, 318 to 322 or 327 to 331 which are known to be important for binding can be used as suitable sites for modification.
- a site capable of forming a disulfide bond such as a site capable of forming a disulfide bond, a few amino acids at the N-terminus in a native Fc, or a methionine residue may be added at the N-terminus of a native Fc.
- complement binding sites such as C1q binding sites may be removed, or ADCC (antibody dependent cell mediated cytotoxicity) sites may be removed to eliminate effector function.
- it may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation and amylation. may be modified.
- the above-described Fc variant is a variant which exhibits the same biological activity as the Fc fragment of the present invention, but increases the structural stability against heat, pH, etc. of the Fc fragment.
- the Fc region may be obtained from natural types separated in vivo from humans and animals such as cows, goats, pigs, mice, rabbits, hamsters, rats, and guinea pigs, and may be obtained from transformed animal cells or microorganisms. It may be recombinant or a derivative thereof.
- the method obtained from the natural form can be obtained by separating the whole immunoglobulin from the human or animal living body, and then treating the protease. Papain is cleaved into Fab and Fc and pepsin is cleaved into pF'c and F (ab) 2 . This may be separated by Fc or pF'c using size-exclusion chromatography.
- the recombinant immunoglobulin Fc region obtained from a microorganism is a human-derived Fc region.
- the immunoglobulin Fc region may be in a natural sugar chain, an increased sugar chain compared to the natural form, a reduced sugar chain or a sugar chain removed from the natural form.
- Conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms can be used to increase or decrease such immunoglobulin Fc sugar chains.
- the immunoglobulin Fc region in which the sugar chain is removed from the Fc has a significant decrease in the binding capacity of the complement (c1q), and the antibody-dependent cytotoxicity or the complement-dependent cytotoxicity is reduced or eliminated, thereby not causing an unnecessary immune response in vivo. Do not.
- a form more consistent with the original purpose as a carrier of the drug would be the immunoglobulin Fc region from which the sugar chains have been removed or unglycosylated.
- the "Deglycosylated" Fc region refers to an Fc region in which an enzyme has been freed of sugar, and the "Aglycosylated” Fc region is produced in a prokaryote, preferably E. coli, and is not glycosylated. Non-Fc fragments.
- the immunoglobulin Fc region may be a human or animal origin, such as cattle, goats, pigs, mice, rabbits, hamsters, rats, guinea pigs, preferably human origin.
- the immunoglobulin Fc region may be an Fc region by IgG, IgA, IgD, IgE, IgM derived or combinations thereof or hybrids thereof. It is preferably derived from IgG or IgM, which is most abundant in human blood and most preferably from IgG known to enhance the half-life of ligand binding proteins.
- “combination” in the present invention means that, when forming a dimer or multimer, a polypeptide encoding a single-chain immunoglobulin Fc fragment of the same origin forms a bond with a single-chain polypeptide of different origin. That is, it is possible to prepare dimers or multimers from two or more fragments selected from the group consisting of Fc fragments of IgG Fc, IgA Fc, IgM Fc, IgD Fc and IgE.
- hybrid is a term that means that within the short-chain immunoglobulin Fc fragment, there is a sequence corresponding to two or more different immunoglobulin Fc fragments of origin.
- various types of hybrids are possible. That is, hybridization of a domain consisting of 1 to 4 domains from the group consisting of CH1, CH2, CH3 and CH4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc and IgD Fc is possible, and may include a hinge.
- IgG can also be divided into subclasses of IgG1, IgG2, IgG3 and IgG4 and combinations or hybridization thereof are also possible in the present invention.
- IgG2 and IgG4 subclasses most specifically the Fc region of IgG4 with little effector function, such as complement dependent cytotoxicity (CDC).
- the immunoglobulin Fc region for a carrier included in the protein conjugate of the present invention may be an unglycosylated Fc fragment derived from human IgG4.
- Human-derived Fc fragments may exhibit superior effects compared to non-human-derived Fc fragments that can cause undesirable immune responses, such as acting as antigens in human living organisms to produce new antibodies against them.
- peptides or protein fragments may be used as carriers to increase half-life in vivo.
- the peptide or protein fragment used may be an Elastin like polypeptide (ELP) consisting of repeating units of a combination of specific amino acids, derivatives thereof such as transferrin or fibronectin, which is a component of connective tissue, which is known to have high stability in vivo.
- ELP Elastin like polypeptide
- fatty acid may be one component that forms a moiety of the conjugate, and means a carboxylic acid of a hydrocarbon chain having one carboxy group (-COOH), R- Monovalent carboxylic acids having the chemical formula of COOH are collectively referred to as fatty acids.
- the fatty acids of the present invention may be saturated fatty acids or unsaturated fatty acids, depending on the bond between the carbon skeletons forming the hydrocarbon chain.
- the unsaturated fatty acid means a fatty acid having one or more double bonds in the bond of the carbon skeleton constituting the hydrocarbon chain, and the fatty acid in which all of the bonds in the carbon skeleton are composed of only a single bond is a saturated fatty acid.
- the fatty acid of the present invention may be a fatty acid having a normal chain structure and may be a fatty acid having a side chain in the alkyl group.
- Fatty acids can be classified into short, medium and long chain fatty acids according to the number of carbons constituting the hydrocarbon chain, generally short-chain fatty acids in the case of having 1 to 6 carbon atoms, medium-chain fatty acids in the case of 6 to 12, long chains of 14 or more May be classified as fatty acids.
- the properties may vary depending on the position of the double bond.
- the "fatty acid derivative" of the present invention may be a substance in which two or more reactors are bonded directly or through a linker to the skeleton of the aforementioned fatty acid.
- the reactor may include 2,5-dioxopyrrolidinyl, 2,5-dioxopyrrolyl, aldehyde, aryldisulfide, heteroaryldisulfide, halogenated acetamide, or C 7-10 alkynyl.
- the reactor is maleimide, N-hydrosuccinimide, succinimide, formaldehyde, acetaldehyde, propionaldehyde, butylaldehyde, orthopyridyl disulfide (OPSS), iodide acetamide, the iodine Instead halogenated acetamide, difluorocyclooctyne (DIFO), dibenzocyclooctyne (DIBO), dibenzo-aza-cyclooctin (Dsibenzo-aza-), including bromine, fluorine, chlorine, or asstatin; cyclooctyne (DIBAC or DBCO), biarylazacyclooctynones (BARAC), tetramethylthiacycloheptin (TMTH), bicyclononyne (BCN) Sondheimer diyne, cyclooctyne OCT), monofluorinated cyclo
- the reactor also binds to the fatty acid backbone via a linker comprising a C 1-3 alkylamino, (C 1-3 alkoxy) n (C 1-3 alkylamino) chain, where n is an integer from 1 to 3.
- linker comprising a C 1-3 alkylamino, (C 1-3 alkoxy) n (C 1-3 alkylamino) chain, where n is an integer from 1 to 3.
- the type of linker is not limited thereto.
- the fatty acid of the present invention is a carboxylic acid having a formula of R-COOH and the corresponding R group may be a linear or branched saturated hydrocarbon group, may be saturated fatty acid or unsaturated fatty acid, may be short, medium or long chain fatty acid, and
- the hydrocarbon chain may be 1 to 40 carbon atoms, more specifically 4 to 30 carbon atoms, but is not limited thereto.
- the fatty acids of the present invention are formic acid (HCOOH, form acid), acetic acid (CH 3 COOH), propionic acid (C 2 H 5 COOH), butyric acid (C 3 H 7 COOH), valeric acid (C 4 H 9 COOH ), Caproic acid (C 5 H 11 COOH), enanthic acid (C 6 H 13 COOH), caprylic acid (C 7 H 15 COOH), pelagonic acid (C 8 H 17 COOH), capric acid (C 9 H 19 COOH), undecyl acid (C 10 H 21 COOH), lauric acid (C 11 H 23 COOH), tridecyl acid (C 12 H 25 COOH), myristic acid (C 13 H 27 COOH), pentade Acid (C 14 H 29 COOH), Palmitic Acid (C 15 H 31 COOH), Heptadecylic Acid (C 16 H 33 COOH), Stearic Acid (C 17 H 35 COOH), Nonadecanoic Acid (C 18 H 37 COOH ), Iraqi acid (HCO
- the fatty acids of the present invention may be derivatives, analogs, and the like of the fatty acids described above, and in particular, the hydrocarbons constituting the fatty acids may be variants containing linear, branched and other ring groups. Ring groups that may be included in the hydrocarbon may be saturated homocycles, heterocycles, aromatic condensed or non-condensed homocycles or heterocycles and may have ether bonds, unsaturated bonds and substituents.
- fatty acid derivatives described in US Patent No. US8129343 and WO2015 / 067715, WO2015 / 055801, WO2013 / 041678, WO2014 / 133324, WO2014 / 009316, WO2015 / 052088 may be used as fatty acids of the present invention. It doesn't work.
- a multi-fatty acid including two or more carboxyl groups, a carboxylic acid (bio) isostere, a substance containing a phosphate group or a sulfonic acid group, a fatty acid ester, and the like may be included without limitation.
- Fatty acid of the present invention may have a molecular weight in the range of 0.1 to 100 kDa, specifically 0.1 to 30 kDa, the fatty acid of the present invention may be used not only one type of polymer but also a combination of different types of polymer, but is not limited thereto. Do not.
- Two or more reactors of the fatty acid derivative included in the protein conjugate of the present invention may be the same or different from each other.
- one reactor may have a maleimide group and the other reactor may have an alkyl aldehyde group such as an aldehyde group, a propion aldehyde group, or butyl aldehyde.
- the conjugate of the present invention can be prepared by using a known chemical reaction to activate the hydroxy group into the various reactors or using a fatty acid having a commercially available modified reactor. Can be.
- a fatty acid is linked to an amino acid residue which is not at the N terminal or the C terminal of the physiologically active polypeptide or the terminal of the polypeptide, and a biocompatible material is connected to the fatty acid moiety linked to the physiologically active polypeptide.
- the N terminus or C terminus comprises a region consisting of 1 to 25 amino acids on the N terminus or C terminus rather than the terminus itself.
- the protein conjugate of the present invention may include one or more structures of [bioactive polypeptide-fatty acid derivative-biocompatible material], and elements constituting the same may be linearly or branched by covalent bonds, and Protein conjugates can include one or more of each component.
- Fatty acid of the present invention includes two or more reactors through which can be covalently linked to the bioactive polypeptide and the biocompatible material, respectively.
- one or more biocompatible polypeptides and fatty acid derivative-linked conjugates are covalently linked to one biocompatible material to form bioactive polypeptide monomers, dimers or multimers via the biocompatible material as a medium. In this way, it is possible to more effectively achieve the increase of in vivo activity and stability of the bioactive polypeptide.
- the bioactive polypeptide and the biocompatible material may be combined in various molar ratios.
- Another embodiment of the present invention comprises the steps of (a) linking a bioactive peptide and a biocompatible material through fatty acid derivatives; And (b) it provides a method for producing a protein conjugate comprising the step of separating the protein conjugate resulting from the reaction of step (a).
- bioactive peptides biocompatible materials and fatty acid derivatives are as described above.
- the connection of the three components may be covalent bonds, the covalent bonds may occur sequentially or simultaneously.
- the connection of the three components may be covalent bonds, the covalent bonds may occur sequentially or simultaneously.
- one of the bioactive polypeptide and the biocompatible material is first bound to one reactor of the fatty acid derivative.
- step (a3) connecting the other one of the biocompatible material and the bioactive polypeptide to the other reactor of the fatty acid derivative of the linker separated in the step (a2), so that the reactor of the fatty acid is biocompatible polypeptide and the biocompatible material, respectively. It may include but is not limited to generating a protein conjugate linked to.
- steps (a1) and (a3) may be carried out in the presence of a reducing agent if necessary in consideration of the type of the reactor of the fatty acid derivative participating in the reaction.
- a reducing agent sodium cyano borohydride (NaCNBH 3 ), sodium borohydride, dimethylamine borate, picoline borane complex or pyridine borate (borane pyridine) may be used.
- Steps (a2) and (b) above may be performed by appropriately selecting among conventional methods used for separation of proteins, considering characteristics such as required purity and molecular weight and charge amount of the resulting product.
- various known methods may be applied, including size exclusion chromatography or ion exchange chromatography, and a plurality of different methods may be used in combination in order to purify to higher purity as needed.
- the reaction molar ratio of the bioactive polypeptide and the fatty acid derivative and the reaction molar ratio of the biocompatible material and the fatty acid derivative may be independently selected from 1: 1 to 1:20.
- the reaction mole ratio of the bioactive polypeptide and the fatty acid derivative may be 1: 2 to 1:10, and the reaction mole ratio of the biocompatible substance and the fatty acid derivative may be 1: 4 to 1:20.
- the reaction molar ratio of the linker separated from (a3) to (a2) and the biocompatible material or physiologically active polypeptide may range from 1: 0.5 to 1:20, but is not limited thereto.
- Another aspect of the present invention provides a protein conjugate prepared by the method of producing a protein conjugate of the present invention and a pharmaceutical composition comprising the protein conjugate prepared above.
- the pharmaceutical composition of the present invention may be a sustained preparation having increased in vivo persistence and stability compared to a native bioactive polypeptide.
- compositions comprising the conjugates of the invention may comprise a pharmaceutically acceptable carrier.
- Pharmaceutically acceptable carriers can be used as oral administration binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments and flavoring agents, and in the case of injectables, buffers, preservatives, analgesic
- a topical agent, a solubilizer, an isotonicity agent, a stabilizer, etc. can be mixed and used, and in case of topical administration, a base, an excipient, a lubricant, a preservative, etc. can be used.
- the formulation of the pharmaceutical composition of the present invention may be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above.
- oral administration may be in the form of tablets, troches, capsules, elixirs, suspensions, syrups and wafers, and in the case of injections, they may be prepared in unit dosage ampoules or multiple dosage forms. And other solutions, suspensions, tablets, pills, capsules, and sustained release preparations.
- suitable carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl Cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil and the like can be used.
- composition of the present invention may further include fillers, anti-coagulants, lubricants, wetting agents, fragrances and preservatives.
- Benzyl 2- (2-hydroxyethoxy) ethylcarbamate (129 g, 0.539 mol) obtained from step 1 was dissolved in THF (2 L), followed by potassium t-butoxide (60.5 g, 0.593 mol). Dropwise at 0 ° C. After 30 minutes, t-butyl bromoacetate was added, stirred at 0 ° C. for 3 hours, and further stirred at room temperature for 15 hours. Water was added to the reaction solution to complete the reaction, followed by concentration, followed by extraction with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated and purified by column chromatography to give the title compound (87.5 g). ) Was obtained.
- Benzyl 2- (2-hydroxyethoxy) ethylcarbamate (15.0 g, 62.691 mmol) obtained in step 1 was dissolved in THF (240 mL), followed by potassium t-butoxide (7.0 g, 62.691 mmol). Dropwise at 0 ° C. After 30 minutes, ethyl bromoacetate was added, stirred at 0 ° C. for 3 hours, and further stirred at room temperature for 15 hours. Water was added to the reaction solution to terminate the reaction, and then concentrated and extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated and purified by column chromatography to obtain the title compound (8.6 g).
- 3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecane-12-oic acid (952 mg, 3.204 mmol) obtained in step 3 was dissolved in acetonitrile (30 mL), BOP ((benzotriazol-1-yloxy) tris (dimethylamino) phosphonium hexafluorophosphate, 1.6 g, 3.522 mmol), N, N-diisopropylethylamine (N, N-diisopropylethylamine; DIPEA, 1.7 mL, 9.606 mmol) Stirred at room temperature for 30 minutes.
- BOP ((benzotriazol-1-yloxy) tris (dimethylamino) phosphonium hexafluorophosphate, 1.6 g, 3.522 mmol)
- N, N-diisopropylethylamine N, N-diisopropylethyl
- An immunoglobulin Fc fragment was used as a biocompatible material of the protein conjugate.
- the immunoglobulin Fc fragment was prepared according to Korean Patent No. 10-0824505, which was filed by the present inventors, in a mass production method of an immunoglobulin Fc region from which a starting methionine residue was removed.
- Example 1 Fatty acid derivative 1- (S) -22- (18- (2- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) comprising two reactors as linker Preparation of ethylamino) -18-oxooctadecaneamido) -1,10,19-trioxo-3,6,12,15-tetraoxa-9,18-diazatricoic acid-23-oic acid
- Example 2 fatty acid derivative 2 comprising two reactors as a linker 2-(S) -22- (18- (2,5-dioxopyrrolidin-1-yloxy) -18-oxooctadecaneamido) -1
- linker 2-(S) -22- (18- (2,5-dioxopyrrolidin-1-yloxy) -18-oxooctadecaneamido
- Example 3 physiological activity with polypeptides Immunoglobulins Fc Preparation of conjugates bound with fatty acid derivative linkers
- the triple activator which is prepared according to Example 1 or 2, having a fatty acid derivative linker comprising a maleimide group and an aldehyde group, exhibiting activity on all GLP-1, GIP, and glucagon receptors, which are a kind of a physiologically active polypeptide (SEQ ID NO:
- the triple activator and the fatty acid derivative linker were mixed at a molar ratio of 1: 1 to 2, and reacted at 4 to 8 ° C. for about 1 to 2 hours.
- the concentration of the triple activator was 3 ⁇ 5 mg / mL
- the reaction was carried out under 20 mM Tris pH 7.0 ⁇ 8.0, isopropanol.
- the reaction solution was purified from triple-activator 1: 1 bound with fatty acid derivative linker using SP-HP (GE, USA) column using citrate (pH 3.0), ethanol-containing buffer and potassium chloride concentration gradient.
- the purified fatty acid derivative linker and the triple activator conjugate and the immunoglobulin Fc fragment were used at a molar ratio of 1: 2-5, and the total protein concentration was 20-35 mg / mL.
- the reaction was carried out for 18 hours.
- the reaction solution was potassium phosphate pH 6.0 ⁇ 6.5, 20 mM sodium cyanoborohydride (SCB) was added as a reducing agent.
- SCB sodium cyanoborohydride
- the reaction solution was subjected to Sourse ISO using a Source Q (GE) column using bis-tris (pH 6.5) buffer and calcium chloride concentration gradient, and a concentration gradient of ammonium sulfate and 20 mM tris (pH 7.5).
- GE Source Q
- the conjugate of triple activator and immunoglobulin Fc covalently linked via a fatty acid derivative linker was purified.
- the prepared protein conjugate was confirmed by SDS-PAGE, and the results are shown in FIG. 1.
- Protein conjugates were prepared using fatty acid derivatives having 1 to 100 carbon atoms that make up the hydrocarbon chain of the fatty acid backbone. Specifically, protein conjugates were prepared using fatty acid derivatives having 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 carbon atoms.
- a protein conjugate prepared according to example 3, specifically, the GLP-1, in vivo (in vivo) pharmacokinetics of both GIP and glucagon receptor conjugate is a triple-active substance and an immunoglobulin Fc represents the activity associated with fatty acid derivatives It was confirmed and compared with the increase in in vivo persistence compared to the conventional triple activator.
- ICR mice widely used as normal animal models were used for pharmacokinetic confirmation. The non-fasting 8-week-old ICR mice were divided into the following two groups after three days of acclimation:
- Group 1 which received triple activators, consisted of 15 individuals, from which 0.3 mL of blood was cross- drawn with orbital veins at 0.25, 0.5, 1, 2 and 4 hours, respectively.
- Group 2 administered a conjugate in which the triple activator of Example 3 and the immunoglobulin Fc were connected through a fatty acid derivative was composed of 12 individuals, and from these individuals 1, 4, 8, 24, 48, 72, At 96, 120, 144 and 168 hours, cross bleeding was performed in the same manner as in group 1. The collected blood was centrifuged (10,000 rpm, 10 minutes, Eppendorf) to separate serum and stored in a freezer at -20 °C.
- the concentration of the triple activator in the serum or the conjugate of the triple activator and the immunoglobulin Fc linked through the fatty acid derivative was quantified by an enzyme-linked immunosorbent assay (ELISA) using an antibody specific for the triple activator.
- ELISA enzyme-linked immunosorbent assay
- Pharmacokinetic parameters using the Phoenix TM WinNonin ® 7.0 (Pharsight, USA) program based on the serum concentration per hour of A, B and C were calculated by non-compartment method (non-compartment method).
- the peak blood concentration (C max ) and time of arrival (T max ) were confirmed by basic data, and the area under the blood drug curve (AUC) over time was calculated by log-linear trapezoidal summation. It was.
- Other pharmacokinetic parameters, such as half- life t 1/2 , distribution volume Vd and clearance CL, were also calculated using the program, and FIG. 2 shows the change in blood concentration of the drug over time.
- the conjugate in which the triple activator and immunoglobulin Fc are linked through a fatty acid derivative according to Example 2 of the present invention has a sustained increase in blood half-life. Indicated.
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Abstract
Description
Claims (25)
- 생리활성 폴리펩타이드 및 생체적합성 물질이 지방산 유도체를 통해 연결된, 단백질 결합체.
- 제1항에 있어서, 상기 생리활성 폴리펩타이드 및 생체적합성 물질이 지방산 유도체와 각각 공유 결합으로 연결된, 단백질 결합체.
- 제1항에 있어서, 상기 지방산 유도체는 지방산 골격에 직접 또는 링커를 통해 연결된 두 개 이상의 반응기를 가지며, 상기 반응기를 통해 생리활성 폴리펩타이드 및 생체적합성 물질과 각각 연결된, 단백질 결합체.
- 제3항에 있어서, 상기 반응기는 2,5-디옥소피롤리디닐, 2,5-디옥소피롤릴, 알데하이드, 아릴디설파이드, 헤테로아릴디설파이드, 할로겐화 아세트아마이드(haloacetamide), 또는 C7-10 알키닐을 포함하는 것인, 단백질 결합체.
- 제3항에 있어서, 상기 반응기는 말레이미드, N-하이드로석신이미드, 석신이미드, 포름알데하이드, 아세트알데하이드, 프로피온알데하이드, 부틸알데하이드, 오르소피리딜 디설파이드(Orthopyridyl disulfide; OPSS), 요오드화 아세트아마이드, 상기 요오드 대신에 브롬, 불소, 염소, 또는 아스타틴을 포함하는 할로겐화 아세트아마이드, 디플루오로사이클로옥틴(difluorocyclooctyne; DIFO), 디벤조사이클로옥틴(dibenzocyclooctyne; DIBO), 디벤조-아자-사이클로옥틴(Dsibenzo-aza-cyclooctyne; DIBAC 또는 DBCO), 바이아릴아자사이클로옥티논(biarylazacyclooctynones; BARAC), 테트라메틸티아사이클로헵틴(tetramethylthiacycloheptyne; TMTH), 바이사이클로노닌(bicyclononyne; BCN) 손드하이머 디인(Sondheimer diyne), 사이클로옥틴(cyclooctyne; OCT), 단불소화 사이클로옥틴(monofluorinated cyclooctyne; MOFO), 디메톡시아자사이클로옥틴(dimethoxyazacyclooctyne; DIMAC), 2,3,6,7-테트라메톡시-디벤조사이클로옥틴(2,3,6,7-tetramethoxy-DIBO, TMDIBO), 술폰화 디벤조사이클로옥틴(sulfonylated DIBO; S-DIBO), 카르복시메틸모노벤조사이클로옥틴(carboxymethylmonobenzocyclooctyne; COMBO), 피롤로사이클로옥틴(pyrrolocyclooctyne; PYRROC) 또는 알카인(alkyne)인 것인, 단백질 결합체.
- 제3항에 있어서, 상기 링커는 C1-3 알킬아미노, (C1-3 알콕시)n(C1-3 알킬아미노) 사슬(여기서, n은 1 내지 3의 정수)을 포함하는 것인, 단백질 결합체.
- 제1항에 있어서, 상기 생체적합성 물질에 생리활성 폴리펩타이드와 지방산 유도체의 연결체가 1개 이상 연결된 것인, 단백질 결합체.
- 제1항에 있어서, 상기 생리활성 폴리펩타이드는 호르몬, 사이토카인, 인터루킨, 인터루킨 결합 단백질, 효소, 항체, 성장인자, 전사조절인자, 혈액인자, 백신, 구조단백질, 리간드 단백질 또는 수용체, 세포표면항원 또는 수용체 길항물질인, 단백질 결합체.
- 제1항에 있어서, 상기 생리활성 폴리펩타이드는 글루카곤 유사 펩타이드-1(GLP-1), 글루카곤, GIP(Gastric inhibitory polypeptide), 옥신토모듈린, 제닌(Xenin), 인슐린, CCK(Cholecystokinin) 아밀린(amylin), 가스트린(gastrin), 그렐린(ghrelin), PYY(peptide YY) 등과 같이 위나 장에서 혈당과 체중을 조절하는 인크레틴류(incretins); 렙틴(Leptin), 아디포넥틴(adiponectin), 아디포린(adipolin), 아페린(apelin), 카르토넥틴(cartonectin)과 같이 지방질(adipose)에서 분비되는 아디포카인류(adipokines); 키스펩틴(Kisspeptin), 네스파틴-1(Nesfatin-1)과 같이 뇌에서 분비되는 뉴로펩타이드류(neuropeptides); 이리신(Irisin), 마이오넥틴(myonectin), 데코린(decorin), 폴리스타틴(follistatin), 머슬린(musclin)과 같이 머슬(muscle)에서 분비되는 펩타이드 혹은 단백질류; 혈관작동성장펩타이드(Vasoactive intestinal peptide), 나트륨이뇨펩타이드류(natriuretic peptides), 호중구 증가 인자(G-CSF), 인간 성장 호르몬(hGH), 에리스로포이에틴(EPO), 성장 호르몬 방출 호르몬, 성장 호르몬 방출 펩타이드, 인터페론, 인터페론 수용체, 지프로테인 관련수용체(G protein-coupled receptor), 인터루킨류, 인터루킨 수용체, 효소류, 인터루킨 결합 단백질, 사이토카인 결합 단백질, 마크로파지 활성인자, 마크로파지 펩타이드, B 세포인자, T 세포인자, 단백질 A, 알러지 억제인자, 세포 괴사 당단백질, 면역독소, 림포독소, 종양 괴사인자, 종양 억제인자, 전이 성장인자, 알파-1 안티트립신, 알부민, α-락트알부민, 아포리포단백질-E, 고 당쇄화 적혈구 생성인자, 안지오포에이틴류, 헤모글로빈, 트롬빈, 트롬빈 수용체 활성 펩타이드, 트롬보모듈린, 혈액인자 VII, VIIa, VIII, IX, 및 XIII, 플라즈미노젠 활성인자, 피브린-결합 펩타이드, 유로키나제, 스트렙토키나제, 히루딘, 단백질 C, C-반응성 단백질, 레닌 억제제, 콜라게나제 억제제, 수퍼옥사이드 디스뮤타제, 혈소판 유래 성장인자, 상피세포 성장인자, 표피세포 성장인자, 안지오스타틴, 안지오텐신, 골 형성 성장인자, 골 형성 촉진 단백질, 칼시토닌, 아트리오펩틴, 연골 유도인자, 엘카토닌, 결합조직 활성인자, 조직인자 경로 억제제, 여포 자극 호르몬, 황체 형성 호르몬, 황체 형성 호르몬 방출 호르몬, 신경 성장인자, 부갑상선 호르몬, 릴랙신, 씨크레틴, 소마토메딘, 인슐린 유사 성장인자, 부신피질 호르몬, 콜레시스토키닌, 췌장 폴리펩타이드, 가스트린 방출 펩타이드, 코티코트로핀 방출인자, 갑상선 자극호르몬, 오토탁신, 락토페린, 미오스타틴, 세포표면항원, 바이러스 유래 백신 항원, 단일클론 항체, 다중클론 항체 및 항체 단편으로 이루어진 군으로부터 선택되는 것인, 단백질 결합체.
- 제1항에 있어서, 상기 생리활성 폴리펩타이드는 2개 이상의 수용체를 동시에 활성화시키는 것을 특징으로 하는 단백질 결합체.
- 제1항에 있어서, 상기 생체적합성 물질은 폴리에틸렌 글리콜(polyethylene glycol, PEG), 콜레스테롤, 알부민 및 이의 단편, 알부민 결합물질, 특정 아미노산 서열의 반복단위의 중합체, 항체, 항체 단편, FcRn 결합물질, 생체 내 결합조직 혹은 그 유도체, 뉴클레오타이드, 파이브로넥틴, 트랜스페린(Transferrin), 사카라이드(saccharide), 고분자 중합체 및 이들의 조합으로 이루어진 군에서 선택된 것인, 단백질 결합체.
- 제11항에 있어서, 상기 FcRn 결합물질은 면역글로불린 Fc 영역을 포함하는 폴리펩타이드인 것인, 단백질 결합체.
- 제12항에 있어서, 상기 면역글로불린 Fc 영역은 비당쇄화된 것인, 단백질 결합체.
- 제12항에 있어서, 상기 면역글로불린 Fc 영역은 힌지(hinge) 영역을 추가로 포함하는 것인, 단백질 결합체.
- 제12항에 있어서, 상기 면역글로불린 Fc 영역은 IgG, IgA, IgD, IgE, IgM, 이들의 조합(combination) 및 이들의 하이브리드(hybrid)로 이루어진 군으로부터 선택되는 것인, 단백질 결합체.
- 제12항에 있어서, 상기 면역글로불린 Fc 영역은 IgG4 Fc 단편인 것인, 단백질 결합체.
- 제1항에 있어서, 상기 지방산 유도체는 탄소수가 1 내지 40인 포화 지방산 또는 불포화 지방산의 유도체인 것인, 단백질 결합체.
- 제17항에 있어서, 상기 지방산은 포름산(HCOOH, form acid), 아세트산(CH3COOH), 프로피온산(C2H5COOH), 부티르산(C3H7COOH), 발레르산(C4H9COOH), 카프로산(C5H11COOH), 에난트산(C6H13COOH), 카프릴산(C7H15COOH), 펠라르곤산(C8H17COOH), 카프르산(C9H19COOH), 운데실산(C10H21COOH), 라우르산(C11H23COOH), 트리데실산(C12H25COOH), 미리스트산(C13H27COOH), 펜타데실산(C14H29COOH), 팔미트산(C15H31COOH), 헵타데실산(C16H33COOH), 스테아르산(C17H35COOH), 노나데칸산(C18H37COOH), 이라크산(C19H39COOH), 베헨산(C21H43COOH), 리그노세르산(C23H47COOH), 세로트산(C25H51COOH), 헵타코산산(C26H53COOH), 몬탄산(C28H57COOH), 멜리스산(C29H59COOH), 락세르산(C31H63COOH), 아크릴산(CH2=CHCOOH), 크로톤산(CH3CH=CHCOOH), 이소크로톤산(CH3CH=CHCOOH), 운데실렌산(CH2=CH(CH2)8COOH), 올레산(C17H33COOH), 엘라이드산(C17H33COOH), 세톨레산(C21H41COOH), 에루크산(C21H41COOH), 브라시드산(C21H41COOH), 소르브산(C5H7COOH, F2), 리놀레산(C17H31COOH, F2), 리놀렌산(C17H29COOH, F3), 아라키돈산(C19H31COOH, F4), 프로피올산(CH≡CCOOH) 및 스테아롤산(C17H31COOH, F1)으로 이루어진 군으로부터 선택된 지방산 또는 이의 유도체인 것인, 단백질 결합체.
- 제18항에 있어서, 상기 지방산은 팔미트산, 미리스트산, 스테아르산 또는 올레산의 지방산 또는 이의 유도체인 것인, 단백질 결합체.
- (a) 생리활성 펩타이드 및 생체적합성 물질을 두 개 이상의 반응기를 갖는 지방산 유도체를 통해 연결하는 단계; 및(b) 상기 (a) 단계의 반응 결과물인 단백질 결합체를 분리하는 단계를 포함하는, 단백질 결합체를 제조하는 방법.
- 제20항에 있어서, 상기 (a) 단계는(a1) 지방산 유도체의 하나의 반응기에 생체적합성 물질과 생리활성 폴리펩타이드 중 어느 하나를 연결시키는 단계;(a2) 상기 (a1) 단계의 반응 혼합물로부터 지방산과 생체적합성 물질과 생리활성 폴리펩타이드 중 어느 하나가 연결된 연결체를 분리하는 단계; 및(a3) 상기 (a2) 단계에서 분리된 연결체의 지방산 유도체의 다른 하나의 반응기에 생체적합성 물질과 생리활성 폴리펩타이드 중 다른 하나를 연결하여, 지방산의 반응기가 각각 생리활성 폴리펩타이드와 생체적합성 물질에 연결된 단백질 결합체를 생성하는 단계를 포함하는, 단백질 결합체를 제조하는 방법.
- 제21항에 있어서, 상기 (a1) 단계 및 (a3) 단계는 환원제의 존재하에서 수행되는 것인, 단백질 결합체를 제조하는 방법.
- 제22항에 있어서, 상기 환원제가 나트륨 시아노 보로하이드라이드(NaCNBH3), 수소화붕소나트륨, 디메틸아민 붕산염, 피콜린 보란 컴플렉스 또는 피리딘 붕산염(borane pyridine)인 것인, 단백질 결합체를 제조하는 방법.
- 제21항에 있어서, 상기 (a1) 단계에서 생리활성 폴리펩타이드와 지방산 유도체의 반응 몰비는 1:1 내지 1:20이고, 생체적합성 물질과 지방산의 반응 몰비는 1:1 내지 1:20인 것인, 단백질 결합체를 제조하는 방법.
- 제21항에 있어서, 상기 (a3) 단계에서 (a2) 단계에서 분리된 연결체와 생체적합성 물질 또는 생리활성 폴리펩타이드의 반응 몰비는 1:0.5 내지 1:20인 것인, 단백질 결합체를 제조하는 방법.
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111164128A (zh) * | 2017-09-29 | 2020-05-15 | 韩美药品株式会社 | 包含作为接头的非肽基聚合物偶联的脂肪酸衍生物化合物的蛋白复合物及其制备方法 |
US11142559B2 (en) | 2016-06-29 | 2021-10-12 | Hanmi Pharm. Co., Ltd. | Glucagon derivative, conjugate thereof, composition comprising same, and therapeutic use thereof |
US11185570B2 (en) | 2017-02-08 | 2021-11-30 | Bristol-Myers Squibb Company | Method of treating cardiovascular disease and heart failure with modified relaxin polypeptides |
US11261227B2 (en) | 2015-06-30 | 2022-03-01 | Hanmi Pharm. Co., Ltd. | Glucagon derivative and a composition comprising a long acting conjugate of the same |
US11332508B2 (en) | 2015-12-31 | 2022-05-17 | Hanmi Pharm. Co., Ltd. | Triple glucagon/GLP-1/GIP receptor agonist |
Families Citing this family (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111406073A (zh) * | 2017-09-29 | 2020-07-10 | 韩美药品株式会社 | 具有提高功效的持久性蛋白质缀合物 |
CN108314621B (zh) * | 2018-04-18 | 2020-09-18 | 斯福瑞(南通)制药有限公司 | 一种制造长链二酸单酯的方法 |
CN111518009B (zh) * | 2019-02-01 | 2023-06-23 | 鲁南制药集团股份有限公司 | 一种脂肪酸衍生物及其合成方法 |
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WO2021050521A1 (en) * | 2019-09-10 | 2021-03-18 | The Broad Institute, Inc. | Compositions, methods and uses for free fatty acid screening of cells at scale |
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KR102352776B1 (ko) * | 2020-02-28 | 2022-01-18 | 아주대학교산학협력단 | 아토피 피부염에 예방 또는 치료 효과가 있는 항염증 펩타이드 |
KR102352775B1 (ko) * | 2020-02-28 | 2022-01-18 | 아주대학교산학협력단 | 아토피 피부염 예방 또는 치료용 신규 펩타이드 |
CN111494641B (zh) * | 2020-04-22 | 2021-08-03 | 南开大学 | 肿瘤微环境响应性的表面电荷可反转纳米药物递送载体 |
EP4265630A1 (en) * | 2020-12-16 | 2023-10-25 | Ajou University Industry-Academic Cooperation Foundation | Anti-inflammatory peptide for preventing or treating atopic dermatitis |
WO2024067401A1 (zh) * | 2022-09-26 | 2024-04-04 | 中美华世通生物医药科技(武汉)股份有限公司 | 包含Fc-高级脂肪酸链的超长效平台 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012087838A1 (en) * | 2010-12-22 | 2012-06-28 | Baxter International Inc. | Materials and methods for conjugating a water soluble fatty acid derivative to a protein |
US8598311B2 (en) * | 2007-11-21 | 2013-12-03 | The University Of The Sciences In Philadelphia | Complexation of fatty acid-conjugated molecules with albumin |
KR20150113934A (ko) * | 2014-03-31 | 2015-10-08 | 한미약품 주식회사 | 면역글로불린 Fc 단편 결합을 이용한 단백질 및 펩타이드의 용해도를 개선시키는 방법 |
Family Cites Families (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5057301A (en) * | 1988-04-06 | 1991-10-15 | Neorx Corporation | Modified cellular substrates used as linkers for increased cell retention of diagnostic and therapeutic agents |
US6307029B1 (en) * | 1996-11-08 | 2001-10-23 | Matsushita Electric Industrial Co., Ltd. | Dye-labeled protein conjugate and method for preparing the same |
ES2252886T3 (es) * | 1998-04-30 | 2006-05-16 | Curis, Inc. | Conjugado de proteinas de erizo activo, proceso para su produccion y uso. |
EP1773878B1 (en) * | 2004-07-19 | 2015-03-04 | Biocon Limited | Insulin-oligomer conjugates, formulations and uses thereof |
US8017151B2 (en) * | 2004-09-07 | 2011-09-13 | Board Of Regents Of The University Of Nebraska By And Behalf Of The University Of Nebraska Medical Center | Amphiphilic polymer-protein conjugates and methods of use thereof |
US8357774B2 (en) * | 2007-09-13 | 2013-01-22 | National University Corporation NARA Institute of Science and Technology | Polypeptide and process for producing the same |
JP5826772B2 (ja) * | 2010-02-16 | 2015-12-02 | ノヴォ ノルディスク アー/エス | コンジュゲートタンパク質 |
KR101382593B1 (ko) * | 2010-07-21 | 2014-04-10 | 한미사이언스 주식회사 | 신규한 지속형 글루카곤 결합체 및 이를 포함하는 비만 예방 및 치료용 약학적 조성물 |
EP2595661A1 (en) * | 2010-07-22 | 2013-05-29 | Novo Nordisk Health Care AG | Growth hormone conjugates |
PT3057984T (pt) * | 2013-10-17 | 2018-10-24 | Boehringer Ingelheim Int | Análogos de glucagon acilados |
-
2016
- 2016-12-02 CN CN201680080891.3A patent/CN109069569B/zh active Active
- 2016-12-02 KR KR1020160163932A patent/KR20170065016A/ko not_active Application Discontinuation
- 2016-12-02 US US15/780,934 patent/US20180360973A1/en not_active Abandoned
- 2016-12-02 EP EP16871097.8A patent/EP3384935A4/en active Pending
- 2016-12-02 JP JP2018528578A patent/JP6989503B2/ja active Active
- 2016-12-02 WO PCT/KR2016/014144 patent/WO2017095201A1/ko active Application Filing
-
2021
- 2021-10-25 JP JP2021173779A patent/JP2022025098A/ja active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8598311B2 (en) * | 2007-11-21 | 2013-12-03 | The University Of The Sciences In Philadelphia | Complexation of fatty acid-conjugated molecules with albumin |
WO2012087838A1 (en) * | 2010-12-22 | 2012-06-28 | Baxter International Inc. | Materials and methods for conjugating a water soluble fatty acid derivative to a protein |
KR20150113934A (ko) * | 2014-03-31 | 2015-10-08 | 한미약품 주식회사 | 면역글로불린 Fc 단편 결합을 이용한 단백질 및 펩타이드의 용해도를 개선시키는 방법 |
Non-Patent Citations (2)
Title |
---|
KLOCKENBUSCH, C. ET AL.: "Optimization of Formaldehyde Cross-linking for Protein Interaction Analysis of Non-tagged Integrin Betal", JOURNAL OF BIOMEDICINE AND BIOTECHNOLOGY, vol. 2010, no. 927585, 2010, pages 1 - 13, XP055387671 * |
KRUGER, S. ET AL.: "Study of the Attachment of Linker Molecules and Their Effects on the Charge Carrier Transfer at Lead Sulfide Nanoparticle Sensitized ZnO Substrates", THE JOURNAL OF PHYSICAL CHEMISTRY C, vol. 115, 2011, pages 13047 - 13055, XP055048979 * |
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US11261227B2 (en) | 2015-06-30 | 2022-03-01 | Hanmi Pharm. Co., Ltd. | Glucagon derivative and a composition comprising a long acting conjugate of the same |
US11667688B2 (en) | 2015-06-30 | 2023-06-06 | Hanmi Pharm. Co., Ltd. | Glucagon derivative and a composition comprising a long acting conjugate of the same |
US11332508B2 (en) | 2015-12-31 | 2022-05-17 | Hanmi Pharm. Co., Ltd. | Triple glucagon/GLP-1/GIP receptor agonist |
US11142559B2 (en) | 2016-06-29 | 2021-10-12 | Hanmi Pharm. Co., Ltd. | Glucagon derivative, conjugate thereof, composition comprising same, and therapeutic use thereof |
US11185570B2 (en) | 2017-02-08 | 2021-11-30 | Bristol-Myers Squibb Company | Method of treating cardiovascular disease and heart failure with modified relaxin polypeptides |
US11364281B2 (en) | 2017-02-08 | 2022-06-21 | Bristol-Myers Squibb Company | Modified relaxin polypeptides comprising a pharmacokinetic enhancer and pharmaceutical compositions thereof |
CN111164128A (zh) * | 2017-09-29 | 2020-05-15 | 韩美药品株式会社 | 包含作为接头的非肽基聚合物偶联的脂肪酸衍生物化合物的蛋白复合物及其制备方法 |
EP3689942A4 (en) * | 2017-09-29 | 2021-06-09 | Hanmi Pharm. Co., Ltd. | PROTEIN CONJUGATE INCLUDING A COMPOUND DERIVED FROM FATTY ACID COUPLED TO A NON-PEPTIDYL POLYMER AS A BOND AND ASSOCIATED PREPARATION PROCESS |
US11357861B2 (en) | 2017-09-29 | 2022-06-14 | Hanmi Pharm. Co., Ltd | Protein complex comprising non-peptidyl polymer-coupled fatty acid derivative compound as linker and preparation method therefor |
Also Published As
Publication number | Publication date |
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US20180360973A1 (en) | 2018-12-20 |
CN109069569B (zh) | 2023-02-14 |
JP2022025098A (ja) | 2022-02-09 |
EP3384935A1 (en) | 2018-10-10 |
EP3384935A4 (en) | 2019-08-21 |
JP6989503B2 (ja) | 2022-01-05 |
KR20170065016A (ko) | 2017-06-12 |
CN109069569A (zh) | 2018-12-21 |
JP2019501144A (ja) | 2019-01-17 |
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