WO2021098720A1 - 抗pcsk9抗体及其应用 - Google Patents

抗pcsk9抗体及其应用 Download PDF

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Publication number
WO2021098720A1
WO2021098720A1 PCT/CN2020/129763 CN2020129763W WO2021098720A1 WO 2021098720 A1 WO2021098720 A1 WO 2021098720A1 CN 2020129763 W CN2020129763 W CN 2020129763W WO 2021098720 A1 WO2021098720 A1 WO 2021098720A1
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Prior art keywords
pcsk9
ldl
hypercholesterolemia
antibody
patient
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PCT/CN2020/129763
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English (en)
French (fr)
Chinese (zh)
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李百勇
夏瑜
王忠民
金小平
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Ad Pharmaceuticals Co Ltd
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Ad Pharmaceuticals Co Ltd
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Priority to MX2022005959A priority Critical patent/MX2022005959A/es
Priority to JP2022528582A priority patent/JP2023501745A/ja
Priority to CA3160071A priority patent/CA3160071A1/en
Priority to BR112022009587A priority patent/BR112022009587A2/pt
Priority to IL292973A priority patent/IL292973B2/en
Priority to IL323016A priority patent/IL323016A/en
Priority to EP20888942.8A priority patent/EP4063396A4/en
Priority to KR1020227020280A priority patent/KR20220107210A/ko
Application filed by Ad Pharmaceuticals Co Ltd filed Critical Ad Pharmaceuticals Co Ltd
Priority to AU2020388182A priority patent/AU2020388182A1/en
Priority to PH1/2022/551172A priority patent/PH12022551172A1/en
Priority to US17/776,295 priority patent/US20230312750A1/en
Publication of WO2021098720A1 publication Critical patent/WO2021098720A1/zh
Anticipated expiration legal-status Critical
Priority to JP2025133754A priority patent/JP2025172265A/ja
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    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
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    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/397—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having four-membered rings, e.g. azetidine
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    • A61K31/33—Heterocyclic compounds
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    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/405—Indole-alkanecarboxylic acids; Derivatives thereof, e.g. tryptophan, indomethacin
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    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
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    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
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    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against enzymes
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    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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    • C07K2317/77—Internalization into the cell
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    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • This application belongs to the field of medical technology and relates to the treatment of hypercholesterolemia. Specifically, this application relates to the use of anti-PCSK9 antibodies to treat hypercholesterolemia.
  • PCSK9 is a secreted serine protease, which binds to the cell surface low-density lipoprotein receptor (LDLR) and is endocytosed and degraded, resulting in a decrease in the number of LDLR on the cell membrane surface.
  • LDLR cell surface low-density lipoprotein receptor
  • PCSK9 is mainly synthesized and secreted by the liver.
  • LDLR is widely distributed on the surface of liver cell membrane and liver cell lines such as HepG2 and Hep3B. Inhibiting the combination of PCSK9 and LDLR can maintain the number of LDLR on the cell membrane surface, thereby mediating plasma cholesterol LDL into cells and maintaining plasma cholesterol levels.
  • Hypercholesterolemia is a disease characterized by defects in lipid metabolism. Some patients carry autosomal dominant genetic mutations called Familial Hypercholesterolemia (FH), which is mainly manifested by low plasma density Lipoprotein cholesterol (LDL-C) levels increased significantly. FH can be divided into four types: heterozygote (Heterozygote), homozygote (Homozygote), compound heterozygote and double heterozygote. The former two types are mostly. Homozygote Familial Hypercholesterolemia (HoFH) is rare, with a prevalence of 1/1 million to 3/1 million. The disease of HoFH is characterized by increased levels of LDL-C and an increased risk of premature atherosclerotic cardiovascular disease.
  • FH Familial Hypercholesterolemia
  • the European Society of Atherosclerosis pointed out that without intervention, HoFH patients usually develop atherosclerotic cardiovascular disease around the age of 20 and die around the age of 30.
  • the main treatment modalities of HoFH include therapeutic lifestyle changes, drug therapy and other treatment modalities, such as plasma lipoprotein replacement.
  • Drug therapy is still dominated by statins, but for patients with HoFH, blood lipid levels are high, and combined treatment with statins and ezetimibe is often required.
  • HeFH heterozygous familial hypercholesterolemia
  • the main treatment modalities of HeFH include therapeutic lifestyle changes, drug therapy and other treatment modalities, such as plasma lipoprotein replacement.
  • Drug therapy is still dominated by statins, but for HeFH patients, blood lipid levels are high, and combined treatment with statins and ezetimibe is often required.
  • PCSK9 inhibitors can directly prevent the combination of PCSK9 and LDLR in the circulation, reduce the decomposition of PCSK9-mediated LDLR, and strengthen its ability to clear LDL-C. On the basis of basic lipid-lowering drugs, it can further reduce the patient's blood lipid level.
  • Alirocumab (formerly known as REGN727/SAR236553) is a fully human PCSK9 monoclonal antibody discovered by the American company Regeneron Pharmaceuticals. In 2015, the FDA approved Alirocumab as a second-line drug for lowering LDL-C. The applicable population is adult patients with hereditary hypercholesterolemia.
  • Cardiovascular disease is one of the main risk factors for death in patients with hyperlipidemia.
  • Current research results suggest that effective lipid-lowering treatments, such as statins, can significantly reduce the risk of cardiovascular disease and death in patients with hyperlipidemia.
  • anti-PCSK9 antibody As an effective hypolipidemic drug, anti-PCSK9 antibody has proved its effectiveness in preventing the onset of cardiovascular disease and reducing the risk of death in patients with hyperlipidemia.
  • the clinical research results of the anti-PCSK9 antibody drug Evolocumab show that patients suffering from hyperlipidemia receiving Evolocumab treatment can significantly reduce the risk of cardiovascular disease in patients with hyperlipidemia. Compared with patients in the placebo control group, patients receiving Evolocumab have myocardial infarction, The incidence of stroke and coronary revascularization was significantly reduced.
  • Alirocumab that has been marketed has also confirmed its protective effect on the cardiovascular system. Compared with the placebo-controlled anti-PCSK9 antibody, it can cause major adverse cardiovascular events, including acute myocardial infarction, ischemic stroke, and Coronary heart disease (CHD) death or unstable angina requiring hospitalization, the overall risk of occurrence is reduced by 15%. Based on the results of such clinical studies, two anti-PCSK9 antibodies were approved by the FDA in 2017 and 2019 to prevent heart disease and stroke in adult patients with cardiovascular disease.
  • One aspect of the present application provides an inhibitor of the interaction between PCSK9 and LDLR, which is preferably an anti-PCSK9 antibody or antigen-binding fragment thereof for the treatment of hypercholesterolemia (more preferably homozygous familial hypercholesterolemia) , Also expressed as "HoFH”, or heterozygous familial hypercholesterolemia, also expressed as "HeFH”, or hypercholesterolemia in patients with extremely high or high cardiovascular risk).
  • hypercholesterolemia more preferably homozygous familial hypercholesterolemia
  • HeFH heterozygous familial hypercholesterolemia
  • the antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof, which specifically binds to human PCSK9 and blocks the binding of human PCSK9 to LDLR.
  • the anti-PCSK9 antibody is a humanized antibody.
  • the anti-PCSK9 antibody comprises:
  • a heavy chain variable region which contains the amino acid sequence shown in SEQ ID NO: 2 or has at least 80% homology, preferably at least 85% homology, more preferably at least 90%, 91% with the amino acid sequence shown in SEQ ID NO: 2 , 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology of amino acid sequences;
  • a light chain variable region which contains the amino acid sequence shown in SEQ ID NO: 4 or has at least 80% homology, preferably at least 85% homology, more preferably at least 90%, 91% with the amino acid sequence shown in SEQ ID NO: 4 , 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology of amino acid sequences.
  • the CDR sequence of the antibody is as follows:
  • HCDR1 GFTFSSYS (SEQ ID NO: 5);
  • HCDR2 ISSSSSYI (SEQ ID NO: 6);
  • HCDR3 EYDFWSAYYDAFDV (SEQ ID NO: 7);
  • LCDR1 SRNIGGGND (SEQ ID NO: 8);
  • LCDR3 QSFDGSLSGSV (SEQ ID NO: 10).
  • compositions preferably for the treatment of hypercholesterolemia (more preferably HoFH, HeFH or hypercholesterolemia in patients with very high or high cardiovascular risk), which comprises a combination of PCSK9 and LDLR Interaction inhibitors (and statins) and pharmaceutically acceptable carriers.
  • the inhibitor of the interaction between PCSK9 and LDLR is in a form suitable for parenteral administration routes or non-parenteral administration routes.
  • kits which is preferably used for the treatment of hypercholesterolemia (more preferably HoFH, HeFH, or hypercholesterolemia in patients with high cardiovascular risk or high risk), which comprises the present invention
  • hypercholesterolemia more preferably HoFH, HeFH, or hypercholesterolemia in patients with high cardiovascular risk or high risk
  • the kit further includes a statin, and/or ezetimibe.
  • the purpose of this application is at least to provide a method for the treatment of hypercholesterolemia (more preferably HoFH, HeFH, or hypercholesterolemia in patients with very high cardiovascular risk or high risk of cardiovascular disease), including administering to subjects in need (For example, parenteral or non-parenteral administration) a therapeutically effective amount of the inhibitor of the interaction between PCSK9 and LDLR of the present invention (preferably an anti-PCSK9 antibody or antigen-binding fragment thereof).
  • the subject is administered a statin in combination. More preferably, the subject is administered a statin and ezetimibe in combination. In some embodiments, the inhibitor, statin, and/or ezetimibe are administered simultaneously or separately.
  • statin is a 3-hydroxy-3 methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor.
  • HMG-CoA 3-hydroxy-3 methylglutaryl coenzyme A
  • statin drugs that is, 3-hydroxy-3 methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors
  • HMG-CoA 3-hydroxy-3 methylglutaryl coenzyme A reductase inhibitors
  • the hypercholesterolemia (more preferably HoFH, HeFH, or hypercholesterolemia in patients with extremely high or high risk of cardiovascular risk) is caused by LDLR, apolipoprotein B (ApoB), PCSK9 Or low density lipoprotein receptor adaptor protein 1 (Low Density Lipoprotein Receptor Adaptor Protein 1 (LDLRAP1)) and other genes caused by one (pair) or more (pair) allelic or non-allelic mutations
  • the allelic mutations include LDLR functional defect mutations, ApoB gene mutations that affect the binding of LDL and LDLR, functional mutations that lead to increased affinity of PCSK9 for LDLR, and LDLRAP1 loss-of-function mutations that lead to decreased LDL internalization activity Wait.
  • the hypercholesterolemia (more preferably HoFH, HeFH, or hypercholesterolemia in patients with a very high or high risk of cardiovascular risk) has LDL-C>13mmol/L without previous treatment (500mg/dL) or (using high-dose/maximum tolerated dose of statins combined with ezetimibe) after treatment with LDL-C ⁇ 8mmol/L (300mg/dL), and at the same time, skin or tendons appear before 10 years of age Xanthomas or both parents carry one (pair) or more (pair) allelic mutations in genes such as LDLR, ApoB, PCSK9, or LDLRAP1.
  • the hypercholesterolemia (more preferably HeFH) satisfies the following a) and b): a) the hypercholesterolemia is derived from LDLR, apolipoprotein B (ApoB) as described above , PCSK9 or Low Density Lipoprotein Receptor Adaptor Protein 1 (LDLRAP1)) and 1 (pair) or more (pair) non-paired allele mutations , B) Serum LDL-C levels in patients who have not received lipid-lowering drug treatment without previous treatment are ⁇ 4.7mmol/L (180mg/dL), or meet at least one of the following c) and d) at the same time: c) Skin/tendon xanthomas or fatty corneal arches in persons under 45 years of age, and d) First-degree relatives with FH or premature adult atherosclerotic cardiovascular disease (ASCVD), especially patients with coronary heart disease.
  • ApoB apolipoprotein B
  • LDLRAP1 Low
  • the purpose of this application is at least to provide an inhibitor of the interaction between PCSK9 and LDLR of the present invention (preferably an anti-PCSK9 antibody or antigen-binding fragment thereof) for preparing treatment of hypercholesterolemia (more preferably HoFH). , HeFH, or hypercholesterolemia in patients with very high cardiovascular risk or high risk of cardiovascular disease).
  • This application also provides a method for treating patients suffering from hypercholesterolemia (more preferably HoFH, HeFH, or hypercholesterolemia in patients with extremely high or high cardiovascular risk), the method comprising: (i) Perform genotyping on the patient, (ii) measure the LDL-C level in the patient’s sample, and (iii) when the patient is identified as homozygous (or heterozygous) familial hypercholesterolemia
  • the patient is given a therapeutically effective amount of the anti-PCSK9 antibody or antigen-binding portion thereof of the present invention, preferably a statin is combined, and more preferably a statin and ezetimibe are combined.
  • the sample is serum, plasma or whole blood.
  • said hypercholesterolemia in patients with very high cardiovascular risk or high-risk risk refers to meeting the following conditions 1 and 2 at the same time, wherein condition 1 meets the conditions in a)-h) Any one (refer to "Guidelines for the Prevention and Treatment of Dyslipidemia in Adults in China (2016 Revised Edition)”): a) History of coronary artery disease (such as acute coronary syndrome, stable coronary heart disease, revascularization, ischemic cardiomyopathy) Etc.), b) History of ischemic stroke (except for lacunar infarction), c) Transient ischemic attack, d) Peripheral atherosclerosis (such as carotid atherosclerosis, after carotid endarterectomy, After carotid stent placement, revascularization, lower extremity atherosclerosis, lower extremity arterial revascularization, etc.), e) LDL-C ⁇ 4.9mmol/L (190mg/dl) or TC ⁇ 7.2 mmol
  • the purpose of this application is at least to provide a method for preparing the interaction between PCSK9 and LDLR for the treatment of hypercholesterolemia (preferably HoFH, HeFH, or hypercholesterolemia in patients with very high cardiovascular risk or high risk of cardiovascular disease)
  • hypercholesterolemia preferably HoFH, HeFH, or hypercholesterolemia in patients with very high cardiovascular risk or high risk of cardiovascular disease
  • Method of pharmaceutical preparation pharmaceutical preparation (pharmaceutical composition) of inhibitor (preferably anti-PCSK9 antibody or antigen-binding fragment thereof).
  • the anti-PCSK9 antibody treatment is administered in a cycle of 2 weeks (14 days) or 4 weeks (28 days), and the anti-PCSK9 antibody is preferably administered subcutaneously on the first day (D1) of each cycle. That is, the anti-PCSK9 antibody is administered at a frequency of once every two weeks (q2w) or once every four weeks (q4w).
  • a single drug dosage unit which contains 0.1-60 mg of the anti-PCSK9 antibody or antigen-binding fragment thereof according to the present invention, preferably for the treatment of familial hypercholesterolemia (more preferably HoFH, HeFH, Or hypercholesterolemia in patients with very high cardiovascular risk or high-risk risk).
  • familial hypercholesterolemia more preferably HoFH, HeFH, Or hypercholesterolemia in patients with very high cardiovascular risk or high-risk risk.
  • the present invention relates to the preparation of 0.1-60 mg of the anti-PCSK9 antibody or antigen-binding fragment thereof according to the present invention for the treatment of familial hypercholesterolemia (more preferably HoFH, HeFH, or with extremely high cardiovascular risk). Or high-risk patients with hypercholesterolemia) in a single drug dosage unit.
  • familial hypercholesterolemia more preferably HoFH, HeFH, or with extremely high cardiovascular risk.
  • high-risk patients with hypercholesterolemia in a single drug dosage unit.
  • MAB1 is an anti-PCSK9 monoclonal antibody, and its sequence and structure can be found in the literature (WO 2016/127912 A1).
  • HCDR1 contains the sequence GFTFSSYS (SEQ ID NO: 5)
  • HCDR2 contains the sequence ISSSSSYI (SEQ ID NO: 6)
  • HCDR3 contains the sequence EYDFWSAYYDAFDV (SEQ ID NO: 7)
  • LCDR1 contains the sequence SRNIGGGND ( SEQ ID NO: 8)
  • LCDR2 includes the sequence GVI (SEQ ID NO: 9)
  • LCDR3 includes the sequence QSFDGSLSGSV (SEQ ID NO: 10).
  • the term "antibody” refers to an antigen binding protein having at least one antigen binding domain.
  • the antibodies and fragments of the present application may be whole antibodies or any fragments thereof. Therefore, the antibodies and fragments thereof of the present application include monoclonal antibodies or fragments thereof, antibody variants or fragments thereof, and immunoconjugates. Antibodies and fragments thereof can also include recombinant polypeptides, fusion proteins, and bispecific antibodies.
  • the anti-PCSK9 antibodies and fragments thereof disclosed herein may be of the IgG1, IgG2, IgG3, or IgG4 isotype.
  • the term "isotype" refers to the antibody species encoded by the heavy chain constant region genes.
  • the anti-PCSK9 antibodies and fragments thereof disclosed herein are of the IgG1 or IgG4 isotype.
  • the anti-PCSK9 antibodies and fragments thereof of the present application can be derived from any species, including but not limited to mice, rats, rabbits, primates, llamas, and humans.
  • the PCSK9 antibody and its fragments can be chimeric antibodies, humanized antibodies or whole human antibodies.
  • humanized antibody refers to an antibody in which the antigen binding site is derived from a non-human species and the variable region framework is derived from human immunoglobulin sequences. Humanized antibodies may contain substitutions in the framework regions, so that the framework may not be an exact copy of the expressed human immunoglobulin or germline gene sequence.
  • gene mutation refers to sudden and heritable mutations in genomic DNA molecules. From a molecular level, gene mutation refers to a change in the base pair composition or arrangement sequence of a gene in the structure. It includes base substitution mutations, frameshift mutations, deletion mutations and insertion mutations.
  • isolated antibody means an antibody that contains substantially no other antibodies with different antigen specificities (for example, an isolated antibody that specifically binds to PCSK9 contains substantially no antibody that specifically binds to antigens other than PCSK9. ). However, an isolated antibody that specifically binds to PCSK9 may have cross-reactivity with other antigens, such as PCSK9 molecules from different species. In addition, the isolated antibody may be substantially free of other cellular materials and/or chemical substances.
  • mAb refers to antibody molecules of single-molecule composition.
  • the monoclonal antibody composition shows a single binding specificity and affinity for a specific epitope, or in the case of a bispecific monoclonal antibody, shows a dual binding specificity for two different epitopes.
  • mAb is an example of an isolated antibody.
  • the mAb can be produced through hybridoma technology, recombinant technology, transgenic technology, or other technologies known to those skilled in the art.
  • antigen-binding portion (also referred to as “antigen-binding fragment”) of an antibody refers to one or more fragments of the antibody that retain the ability to specifically bind to the antigen bound by the intact antibody.
  • antigen-binding fragments include Fab fragments, Fab' fragments, F(ab)' fragments, Fv fragments, isolated CDR regions, single-chain Fv molecules (scFv), F(ab') 2 , Fd, dAb, Fab/c , Bivalent antibodies and domain antibodies.
  • the term "derived" when used to refer to a molecule or polypeptide relative to a reference antibody or other binding protein means a molecule or polypeptide capable of specifically binding to the same epitope as the reference antibody or other binding protein.
  • EC50 refers to the effective concentration, 50% of the maximum response of the antibody.
  • IC50 refers to the inhibitory concentration, 50% of the maximum response of an antibody. Both EC50 and IC50 can be measured by ELISA or FACS analysis or any other method known in the art.
  • treatment generally refers to an operation to obtain a desired pharmacological and/or physiological effect.
  • the effect may be prophylactic in terms of completely or partially preventing the disease or its symptoms; and/or may be therapeutic in terms of partially or completely stabilizing or curing the disease and/or side effects due to the disease.
  • Treatment covers any treatment of a patient's disease, including: (a) preventing diseases or symptoms in patients who are susceptible to diseases or symptoms but have not yet been diagnosed with the disease; (b) suppressing the symptoms of the disease, That is to prevent its development; or (c) alleviate the symptoms of the disease, that is, cause the disease or symptoms to degenerate.
  • systemic therapy refers to a treatment in which a drug substance is transported through the bloodstream to reach and affect cells throughout the body.
  • the term "subject” refers to mammals, such as rodents, felines, canines, and primates.
  • the subject according to the present application is a human.
  • administering means using any of a variety of methods and delivery systems known to those skilled in the art to physically introduce a composition containing a therapeutic agent to a subject.
  • the route of administration of immune checkpoint inhibitors includes intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion.
  • parenteral administration refers to modes of administration other than enteral and local administration that are usually performed by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, and intralymphatic , Intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspine, epidural and intrasternal injections and infusions , And electroporation in vivo.
  • the immune checkpoint inhibitor (e.g., anti-PCSK9 antibody) is administered by a non-parenteral route, and in certain embodiments, it is administered orally.
  • non-parenteral routes include topical, epidermal or mucosal routes of administration, for example, intranasal, vaginal, rectal, sublingual, or topical. Administration can also be performed, for example, one time, multiple times, and/or over one or more extended periods of time.
  • an "adverse event” as used herein is any unfavorable and usually unintentional or undesirable sign (including abnormal laboratory findings), symptom, or disease related to the application of medical treatment.
  • an adverse event may be related to a liver that secretes PCSK9 in response to treatment.
  • Medical treatments can have one or more related AEs, and each AE can have the same or different severity levels.
  • the reference to a method capable of "changing adverse events” refers to a treatment plan that reduces the incidence and/or severity of one or more AEs related to the application of different treatment plans.
  • Dosing interval refers to the amount of time that elapses between multiple doses of the formulations disclosed herein that are administered to a subject. Thus, the dosing interval can be indicated as a range.
  • dosing frequency refers to the frequency with which the dosage of the formulation disclosed herein is administered at a given time.
  • the frequency of dosing can be indicated as the number of dosing at a given time, for example, once a week or once every two weeks.
  • flat dose refers to the dose administered to a patient regardless of the weight or body surface area (BSA) of the patient. Therefore, the uniform dose is specified as the absolute amount of the drug (for example, anti-PCSK9 antibody), rather than being specified as the mg/kg dose. For example, a 60 kg person and a 100 kg person will receive the same dose of antibody (e.g., 240 mg of anti-PCSK9 antibody).
  • BSA body surface area
  • fixed dose means that two or more different antibodies in a single composition are present in the composition in a specific (fixed) ratio to each other.
  • the fixed dose is based on the weight of the antibody (e.g., mg).
  • the fixed dose is based on the concentration of the antibody (e.g., mg/ml).
  • the ratio of the first antibody (mg): second antibody (mg) is at least about 1:1, about 1:2, about 1:3, about 1:4, about 1:5 , About 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, about 1:30, about 1:40, about 1:50 , About 1:60, about 1:70, about 1:80, about 1:90, about 1:100, about 1:120, about 1:140, about 1:160, about 1:180, about 1:200 , About 200:1, about 180:1, about 160:1, about 140:1, about 120:1, about 100:1, about 90:1, about 80:1, about 70:1, about 60:1 , About 50:1, about 40:1, about 30:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1 , About 5:1, about 4:1, about 3:1 or about 2:1.
  • a ratio of 3:1 between the first antibody and the second antibody may mean that the bottle may
  • Patient includes any human or non-human animal.
  • non-human animal includes, but is not limited to, vertebrates such as non-human primates, sheep, dogs, and rodents such as mice, rats, and guinea pigs.
  • the patient is a human.
  • subject and patient are used interchangeably in certain contexts herein.
  • a “therapeutically effective amount” or “therapeutically effective dose” of a drug or therapeutic agent is any amount of a drug that protects patients from disease onset or promotes disease regression when used alone or in combination with another therapeutic agent.
  • the regression of the disease is evidenced by a reduction in the severity of the disease symptoms, an increase in the frequency and duration of the symptom-free phase, or the prevention of injury or disability caused by the torture of the disease.
  • the ability of therapeutic agents to promote disease regression can be evaluated using a variety of methods known to skilled practitioners, such as in human patients during clinical trials, in animal model systems that predict efficacy in humans, or by in vitro assays The activity of the agent is determined in the.
  • the term “about”, “approximately” or “substantially comprising” means a value or composition within the acceptable error range of a particular value or composition determined by a person of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. Composition, that is, the limits of the measurement system. For example, “about”, “approximately” or “substantially comprising” can mean within 1 or more than 1 standard deviation according to practice in the art. Alternatively, “about” or “substantially comprising” may refer to a range that differs from the parameter or value modified by it by at most 10% or 20% (ie, ⁇ 10% or ⁇ 20%).
  • about 3 mg can include any number between 2.7 mg and 3.3 mg (for 10%) or between 2.4 mg and 3.6 mg (for 20%).
  • the term can mean up to an order of magnitude or up to 5 times the value.
  • a dosing interval of about once every 6 weeks or about once every 12 weeks means that the first dose can be administered on any day of the first week, and then the first dose can be administered in the sixth week or the twelfth week, respectively.
  • the second dose is administered any day.
  • a dosing interval of about once every 6 weeks or about once every 12 weeks means that the first dose is administered on a specific day (e.g., Monday) of the first week, and then on the sixth week or the first dose, respectively.
  • the second dose was administered on the same day of the twelve weeks (i.e., Monday). Similar principles apply to phrases including, but not limited to, "approximately once every 2 weeks", “approximately once a month”, etc...
  • any concentration range, percentage range, ratio range, or integer range should be understood to include the value of any integer within the recited range, and where appropriate, a fraction thereof (such as one-tenth of an integer) And one percent) unless otherwise indicated.
  • a pH of about 5.5 means a pH of 5.5 ⁇ 5%, preferably a pH of 5.5 ⁇ 2%, and more preferably a pH of 5.5 ⁇ 1%.
  • pharmaceutically acceptable refers to those compounds, materials, compositions and/or dosage forms that are within the scope of reliable medical judgment and are suitable for use in contact with human and animal tissues without excessive Toxicity, irritation, allergic reactions or other problems or complications of the disease are commensurate with a reasonable benefit/risk ratio.
  • pharmaceutical preparation and “pharmaceutical composition” refer to a mixture of an active ingredient of the present application (for example, an anti-PCSK9 antibody) and pharmaceutically acceptable excipients.
  • active ingredient of the present application for example, an anti-PCSK9 antibody
  • pharmaceutically acceptable excipients for example, an anti-PCSK9 antibody
  • the purpose of the pharmaceutical composition is to facilitate the administration of the active ingredients of the present application to the subject.
  • single drug dosage unit means a single dose containing the anti-PCSK9 antibody or antigen-binding fragment thereof of the present invention to be administered to the subject at the time of the dosing schedule (preferably per kg body weight of the subject)
  • Pharmaceutical dosage forms such as ampoules.
  • the following content does not limit the administration method of the drug of the application.
  • the medicament of the present application can be formulated into a pharmaceutical composition suitable for single or multiple administration.
  • the drugs of the present application are administered by various suitable routes, including, but not limited to, oral or parenteral (by intravenous, intramuscular, topical or subcutaneous routes).
  • the medicament of the present application is administered orally or by injection, such as intravenous injection or intraperitoneal injection.
  • Suitable dosage forms of the medicine of the present application include, but are not limited to, tablets, troches, pills, capsules (such as hard capsules, soft capsules, enteric-coated capsules, microcapsules), elixirs, granules, syrups, injections (Intramuscular, intravenous, intraperitoneal), granules, emulsions, suspensions, solutions, dispersions, and dosage forms of sustained-release preparations for oral or parenteral administration.
  • the pharmaceutical composition of the present application contains pharmaceutically acceptable carriers and/or excipients.
  • Figure 2 The effect of MAB1 on serum LDL-C of cynomolgus monkeys, s.c: subcutaneous injection; i.v: intravenous injection; male: male; female: female.
  • the isotype control antibody is human anti-Hen Egg Lysozyme IgG (anti-HEL, namely human IgG, hIgG for short), and its sequence is derived from Affinity published by Acierno et al. maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies (Acierno et al. J Mol Biol. 2007; 374(1): 130-46.
  • the amino acid sequence of the heavy chain is shown in SEQ ID NO: 11
  • the amino acid sequence of the light chain is shown in SEQ ID NO: 12
  • the isotype control antibody designed for this study was prepared in the laboratory of Zhongshan Kangfang Biomedical Co., Ltd.
  • mice used were purchased from Guangdong Medical Experimental Animal Center.
  • the present inventors artificially designed a series of antibody sequences based on the existing PCSK9 protein sequence (NP_777596.2) and its three-dimensional crystal structure. Through a large number of screening and testing, a MAB1 that specifically binds to PCSK9 was finally obtained.
  • the amino acid sequences of the heavy chain and light chain variable regions of the monoclonal antibody and their coding sequences are shown in the following SEQ ID NO: 1-4.
  • the designed DNA sequence of MAB1 heavy chain VH is as follows (369bp):
  • VH protein sequence is as follows (123aa):
  • the DNA sequence of MAB1 light chain VL is as follows (333bp):
  • the encoded VL protein sequence is as follows (111aa):
  • the CDR sequence of the antibody is as follows:
  • HCDR1 GFTFSSYS (SEQ ID NO: 5);
  • HCDR2 ISSSSSYI (SEQ ID NO: 6);
  • HCDR3 EYDFWSAYYDAFDV (SEQ ID NO: 7);
  • LCDR1 SRNIGGGND (SEQ ID NO: 8);
  • LCDR3 QSFDGSLSGSV (SEQ ID NO: 10).
  • the heavy chain cDNA sequence of MAB1 (the coding sequence is shown in SEQ ID NO: 1; the constant region is Ig gamma-1 chain C region, ACCESSION: P01857) and the cDNA sequence of the light chain (the coding sequence is shown in SEQ ID NO: 3) Show; constant regions are Ig lambda-2 chain C regions; ACCESSION: POCG05.1) were cloned into pUC57simple (provided by GenScript) vector to obtain pUC57simple-MAB1H and pUC57simple-MAB1L plasmids.
  • the plasmids pUC57simple-MAB1H and pUC57simple-MAB1L were digested (HindIII&EcoRI), and the heavy and light chains recovered by electrophoresis were subcloned into pcDNA3.1 vector, and the recombinant plasmids were extracted and co-transfected into 293F cells. After the cells were cultured for 7 days, the culture solution was centrifuged at a high speed, the supernatant was concentrated, and then loaded onto the HiTrap MabSelect SuRe column. The protein was eluted in one step with Elution Buffer and the target sample was recovered and the medium was changed to PBS.
  • mice 32 C57BL/6 mice were collected from orbital blood to separate serum before administration, and the low-density lipoprotein cholesterol (LDL-C) of mice was detected using an automatic biochemical analyzer (Mindray, model BS-180).
  • LDL-C low-density lipoprotein cholesterol
  • Table 1 the mice were divided into 4 groups, namely the normal group, the model group, the MAB1 low-dose group and the MAB1 high-dose group, with 8 mice in each group, and they were administered according to the dosage regimen in Table 1. And before administration D0, D3, D7, D10, D14, D18 after administration, blood was collected to separate serum, and blood lipid and low-density lipoprotein cholesterol were detected.
  • the first time of administration is D0, that is, day 0; subcutaneous: that is subcutaneous injection;
  • the computer system was used to randomly assign the animals to the MAB1 low-dose group, MAB1 medium-dose group, MAB1 high-dose group and MAB1 intravenous administration group according to Table 2.
  • Dosing schedule administration And in all animals at D0 before the drug, 4h after the drug, D2, D4, D6, D8, D11, D15, D18, D22, about 0.5ml blood samples were collected on an empty stomach. After the blood sample is collected, it is transferred to a centrifuge tube and centrifuged at 2-8°C and 3000g for 10 minutes. Determination of low-density lipoprotein (LDL-C).
  • LDL-C low-density lipoprotein
  • the first administration time is D0, that is day 0; subcutaneous: that is subcutaneous injection; intravenous; that is intravenous injection; according to the animal body weight measured before the administration, the animals are randomly assigned to 4 groups by sex using a computer system . Each group has 3 females and 3 males;
  • PCSK9 can inhibit the expression of LDLR on the surface of HepG2 cells by mediating the endocytosis of LDLR.
  • LDL can bind to LDLR and then be endocytosed into cells.
  • BODIPY fluorescent-labeled LDL (BODIPY-LDL, Life, Item No.: L3483) can be used as a marker to bind to LDLR and be endocytosed into cells. By detecting the fluorescence value of BODIPY-LDL in the cell , which in turn reflects the endocytosis of LDL mediated by LDLR.
  • PCSK9 exists and binds to LDLR, it will reduce the LDLR on the cell surface and decrease the BODIPY-LDL bound to LDLR, that is, inhibit the endocytosis of LDL; conversely, inhibiting PCSK9 can enhance the endocytosis of LDL by HepG2 cells. Therefore, this experimental system can measure the pharmacological activity of anti-PCSK9 antibodies by detecting the endocytosis activity of HepG2 cells to BODIPY-LDL.
  • Pre-dilute PCSK9 to 6000 nM with analysis medium (1% NEAA DMEM), add 10 ⁇ l to each well; dilute the antibody to 10 ⁇ , add 10 ⁇ l to each well, and place it in a 37°C, 5% CO 2 incubator for 6 hours. After that, 10 ⁇ l of 0.1 mg/ml BODIPY-LDL solution was added to each well and placed in a 37°C, 5% CO 2 incubator for 22 hours. Take out the culture plate, discard the culture medium, add 200 ⁇ l PBS to each well and discard after washing. Add formaldehyde solution to fix the cells and place them at room temperature for 20 minutes. Discard the formaldehyde and add PBS to wash.
  • analysis medium 1% NEAA DMEM
  • the EC50 of MAB1 is 12.99 ⁇ g/ml.
  • PCSK9 There are many types of mutations in PCSK9. According to the different effects of mutations on PCSK9's LDL-C level, it can be divided into two types, gain-of-function and loss-of-function. Gain-of-function mutations can enhance the ability of PCSK9 to up-regulate LDL-C, leading to hypercholesterolemia.
  • the main forms are S127R, D129G, F216L, R218S, D374Y, N425S, R496W, H553R, and E670G.
  • the D374Y (Asp374Tyr) mutation changes the affinity of PCSK9 and LDLR, which increases the affinity of PCSK9 and LDLR by 5 to 30 times (Lagace TA et al. J Clin Invest. 2006, 116(11): 2995-3005; Cunningham D et al. Nat Struct Mol Biol, 2007, 14(5): 413-419; Fisher TS et al. J Biol Chem, 2007, 282(28): 20502-20512).
  • the D374Y (Asp374Tyr) mutant protein was used as an example to study the activity of MAB1 to inhibit the endocytosis of LDLR mediated by the enhanced PCSK9 mutant protein.
  • the experimental results suggest that MAB1 can effectively relieve the inhibition of the endocytosis of LDL by the gain-of-function PCSK9 protein.
  • HepG2 purchased from the Chinese Academy of Sciences, P14
  • DMEM Gibco, article number: 11995040
  • FBS Excell Bio, article number: FSP500
  • MEM NEAA 100X
  • POLY-L -LYSINE SIGMA, article number: P4832
  • BODIPY-LDL Life, article number: L3483
  • PCSK9-his produced by Kangfang Biotechnology
  • PCSK9 (D374Y)-his Acrobio, article number: PCY-H5225
  • Evolocumab available from AMGEN).
  • PCSK9 and antibody were diluted with assay medium (final concentration as shown in the figure), and added to a 96-well plate, the final volume of the system was 100 ⁇ L.
  • Remove the culture plate add 10 ⁇ L of 0.1 mg/mL BODIPY-labeled LDL solution to each well, and place the culture plate at 37° C. and 5% CO 2 for 9 hours.
  • REU relative fluorescence units
  • Example 4 relieves wild-type PCSK9 protein from the inhibition of mutant LDLR-mediated LDL endocytosis
  • PCSK9 protein binds to LDLR to regulate the metabolism of LDL-C.
  • LDLR gene mutations There are many LDLR gene mutations. Based on LDLR protein synthesis and function, mutations can be divided into 1) LDLR non-expressing mutations, including non-expression of LDLR alleles, that is, cells that do not express LDLR, specific promoter sequence mutations and nonsense mutations , Frameshift mutations, splicing mutations, etc., as well as LDLR transport defects, that is, defective LDLR cannot be transported from the endoplasmic reticulum to the Golgi apparatus after synthesis and then accumulated in the cell to be decomposed; 2) Combination-defective gene mutations, that is, the mutant gene codes The abnormal LDLR can be transported and expressed on the cell surface, but the activity of binding to LDL-C decreases or disappears; 3) Inward transfer defective gene mutation, that is, due to mutations in the codon encoding the NPVY sequence, the receptor can bind
  • the wild-type LDLR that is, the amino acid at position 227 corresponding to the following mutant LDLR (D227E) is D
  • the mutant LDLR (D227E) (https://www.ncbi.nlm.nih.gov/clinvar/variation/ 3690/) as a control to evaluate the endocytosis activity of different mutant LDLRs, the inhibition of PCSK9 on the endocytosis of different mutant LDLRs, and whether MAB1 effectively relieves the inhibition of PCSK9-mediated LDL endocytosis.
  • Plasmid and PEI (Polyscience, catalog number: 23966), target plasmid and control plasmid (constructed by Zhongshan Kangfang Biomedicine Co., Ltd., see Table 5) (each 2 ⁇ g, PEI 10 ⁇ g (single-transformed PEI dosage 5 ⁇ g), diluted DNA Mix and incubate with PEI for 15 minutes, then slowly drip into the 6-well plate, shake gently, and incubate overnight in the incubator; change the medium in the 6-well plate to 2mL DMEM+10% FBS, and place it in the incubator to continue culturing ; 48h after transfection, 0.05% trypsin-EDTA digestion and centrifugation to collect cells, count; take some cells for reporter assay experiment, 2*10 5 /well, 100 ⁇ L DMEM+10% FBS, PBS and IL-6 (Kangfang Bio , Lot number: 20150421) (50ng/mL) group, after incubating overnight, add 50 ⁇ L of substrate (Bright-Gl

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2025522576A (ja) * 2022-06-30 2025-07-15 エーディー ファーマシューティカルズ カンパニー，リミティド 抗ｐｃｓｋ９抗体製剤及びその応用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102232088A (zh) * 2007-10-26 2011-11-02 先灵公司 用于治疗脂类和胆固醇疾病的抗pcsk9及方法
US20140099312A1 (en) * 2008-12-15 2014-04-10 Regeneron Pharmaceuticals, Inc. Methods for Treating Hypercholesterolemia and Reducing LDL-C Using Antibodies to PCSK9
CN104583237A (zh) * 2012-05-08 2015-04-29 奥尔德生物控股有限责任公司 抗pcsk9抗体及其用途
CN105263963A (zh) * 2013-05-30 2016-01-20 瑞泽恩制药公司 伴pcsk9功能获得性突变的常染色体显性高胆固醇血症的治疗方法
CN105461809A (zh) * 2015-02-11 2016-04-06 中山康方生物医药有限公司 Pcsk9抗体、其药物组合物及其用途
CN107106678A (zh) * 2014-07-16 2017-08-29 赛诺菲生物技术公司 用于治疗患有高胆固醇血症的高心血管风险患者的方法

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JOP20080381B1 (ar) * 2007-08-23 2023-03-28 Amgen Inc بروتينات مرتبطة بمولدات مضادات تتفاعل مع بروبروتين كونفيرتاز سيتيليزين ككسين من النوع 9 (pcsk9)
JOP20200043A1 (ar) * 2011-05-10 2017-06-16 Amgen Inc طرق معالجة أو منع الاضطرابات المختصة بالكوليسترول
EA039663B1 (ru) * 2012-05-03 2022-02-24 Амген Инк. Применение антитела против pcsk9 для снижения сывороточного холестерина лпнп и лечения связанных с холестерином расстройств
ES2914978T3 (es) * 2013-10-11 2022-06-20 Sanofi Biotechnology Uso de un inhibidor de PCSK9 para tratar hiperlipidemia
KR20230074283A (ko) * 2014-07-16 2023-05-26 사노피 바이오테크놀로지 이형접합성 가족성 고콜레스테롤혈증(heFH) 환자의 치료방법
EA039310B1 (ru) * 2015-02-26 2022-01-12 Санофи Байотекнолоджи Способы снижения сердечно-сосудистого риска
CN108239150A (zh) * 2016-12-24 2018-07-03 信达生物制药(苏州)有限公司 抗pcsk9抗体及其用途

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102232088A (zh) * 2007-10-26 2011-11-02 先灵公司 用于治疗脂类和胆固醇疾病的抗pcsk9及方法
US20140099312A1 (en) * 2008-12-15 2014-04-10 Regeneron Pharmaceuticals, Inc. Methods for Treating Hypercholesterolemia and Reducing LDL-C Using Antibodies to PCSK9
CN104583237A (zh) * 2012-05-08 2015-04-29 奥尔德生物控股有限责任公司 抗pcsk9抗体及其用途
CN105263963A (zh) * 2013-05-30 2016-01-20 瑞泽恩制药公司 伴pcsk9功能获得性突变的常染色体显性高胆固醇血症的治疗方法
CN107106678A (zh) * 2014-07-16 2017-08-29 赛诺菲生物技术公司 用于治疗患有高胆固醇血症的高心血管风险患者的方法
CN105461809A (zh) * 2015-02-11 2016-04-06 中山康方生物医药有限公司 Pcsk9抗体、其药物组合物及其用途
WO2016127912A1 (zh) 2015-02-11 2016-08-18 中山康方生物医药有限公司 Pcsk9抗体、其药物组合物及其用途

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
"Guidelines for Prevention and Treatment of Dyslipidemia in Chinese Adults", 2016
ACIERNO ET AL., JMOL BIOL, vol. 374, no. 1, 2007, pages 130 - 46
CANADIAN JOURNAL OF CARDIOLOGY, vol. 34, 2018, pages 1553 - 1563
CUNNINGHAM D ET AL., NAT. STRUCT. MOL. BIOL., vol. 14, no. 5, 2007, pages 413 - 419
FISHER TS ET AL., J. BIOL. CHEM., vol. 282, no. 28, 2007, pages 20502 - 20512
LAGACE TA ET AL., J. CLIN. INVEST., vol. 116, no. 11, 2006, pages 2995 - 3005
LIANG L ET AL., SCIENTIFIC REPORTS, vol. 5, no. 1, 2015, pages 17272
See also references of EP4063396A4

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2025522576A (ja) * 2022-06-30 2025-07-15 エーディー ファーマシューティカルズ カンパニー，リミティド 抗ｐｃｓｋ９抗体製剤及びその応用

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