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)
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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 BR112022009587A priority Critical patent/BR112022009587A2/pt
Priority to AU2020388182A priority patent/AU2020388182A1/en
Priority to MX2022005959A priority patent/MX2022005959A/es
Priority to CA3160071A priority patent/CA3160071A1/en
Priority to KR1020227020280A priority patent/KR20220107210A/ko
Priority to IL323016A priority patent/IL323016A/en
Priority to PH1/2022/551172A priority patent/PH12022551172A1/en
Priority to EP20888942.8A priority patent/EP4063396A4/en
Application filed by Ad Pharmaceuticals Co Ltd filed Critical Ad Pharmaceuticals Co Ltd
Priority to IL292973A priority patent/IL292973B2/en
Priority to US17/776,295 priority patent/US20230312750A1/en
Priority to JP2022528582A priority patent/JP2023501745A/ja
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
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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
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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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    • 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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    • A61K31/40—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
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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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    • A61K31/495—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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    • 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
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    • 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
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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
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    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
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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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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
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    • 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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Abstract

提供了抗PCSK9抗体及其应用，优选用于治疗高胆固醇血症。

Description

抗PCSK9抗体及其应用 技术领域
本申请属于医药技术领域，涉及可用于高胆固醇血症的治疗。具体的，本申请涉及抗PCSK9抗体的治疗高胆固醇血症的用途。
背景技术
PCSK9是一种分泌型丝氨酸蛋白酶，可与细胞表面低密度脂蛋白受体(Low-density lipoprotein receptor，LDLR)结合并内吞降解，从而导致细胞膜表面LDLR数目减少，PCSK9主要由肝脏合成并分泌释放至血液。LDLR广泛分布于肝脏细胞膜表面以及肝脏细胞系如HepG2、Hep3B表面。抑制PCSK9与LDLR的结合，可以保持细胞膜表面LDLR数目，进而介导血浆胆固醇的LDL进入细胞，维持血浆胆固醇水平。
高胆固醇血症是一种以脂质代谢缺陷为特征的疾病，其中某些患者携带有常染色体显性遗传突变称为家族性高胆固醇血症(Familial Hypercholesterolemia，FH)，主要表现为血浆低密度脂蛋白胆固醇(LDL-C)水平显著升高。FH可分为杂合子(型)(Heterozygote)、纯合子(型)(Homozygote)、复合杂合子和双重杂合子4种类型，以前两类居多。纯合子家族性高胆固醇血症(Homozygote Familial Hypercholesterolemia，HoFH)罕见，患病率为1/100万～3/100万。HoFH的疾病特点是LDL-C水平升高和早发动脉粥样硬化性心血管疾病风险升高。欧洲动脉粥样硬化学会(EAS)指出，若不加以干预，HoFH患者通常于20岁左右发生动脉粥样硬化性心血管疾病，30岁左右死亡。HoFH的主要治疗方式包括治疗性生活方式改变、药物治疗和其他治疗方式，比如血浆脂蛋白置换。药物治疗仍以他汀类为主，但是对于HoFH患者，血脂水平较高，常常需要他汀类与依折麦布联合治疗。
杂合子(型)家族性高胆固醇血症(HeFH)的患病率约为1∶500～1∶200，全球范围内约有1400～3400万的HeFH患者。HeFH的主要治疗方式包括治疗性生活方式改变、药物治疗和其他治疗方式，比如血浆脂蛋白置换。 药物治疗仍以他汀类为主，但是对于HeFH患者，血脂水平较高，常常需要他汀类与依折麦布联合治疗。
PCSK9抑制剂能直接阻止循环中PCSK9与LDLR相结合，减少PCSK9介导的LDLR的分解，加强其对LDL-C的清除能力，在基础降脂药物基础上，能进一步降低患者血脂水平。目前已经上市的靶向于PCSK9单克隆抗体有两个：Alirocumab和Evolocumab。Alirocumab(曾用名REGN727/SAR236553)由美国公司Regeneron Pharmaceuticals发现的全人源PCSK9单克隆抗体，2015年FDA批准Alirocumab作为二线药物用于降低LDL-C，适用人群为遗传性高胆固醇血症成年患者以及饮食干预和他汀类药物无效而需要降低LDL-C的动脉粥样硬化成年患者。在2015年08月27日，美国食品药品监督管理局(FDA)批准依洛尤单抗(Evolocumab)注射剂用于HoFH患者、及临床动脉粥样硬化心血管疾病的成人患者，在饮食控制和最大耐受剂量他汀类药物治疗的基础上，联合使用Evolocumab来进行治疗。2018年07月31日，中国国家药品监督管理局(NMPA)批准Evolocumab注射液用于治疗成人或12岁以上青少年HoFH(即纯合子家族性高胆固醇血症)。
心血管疾病是高血脂患者的主要死亡风险因素之一，目前研究结果提示有效的降血脂治疗，如他汀类，可显著降低高血脂患者的心血管疾病发作及死亡风险。抗PCSK9抗体作为一种有效的降血脂药物，其在预防高血脂患者心血管疾病发作和降低死亡风险中的有效性亦得到了证实。抗PCSK9抗体药物Evolocumab的临床研究结果显示证实，罹患高血脂的患者接受Evolocumab治疗可显著降低高血脂患者心血管疾病发作的风险，相较于安慰剂对照组患者，接受Evolocumab治疗患者的心肌梗死、卒中及接受冠状动脉血运重建术的发生率显著降低。另一已上市抗PCSK9抗体Alirocumab的大规模临床研究亦证实其对心血管的保护作用，相较于安慰剂对照抗PCSK9抗体可使主要不良心血管事件，包括急性心肌梗死、缺血性卒中、冠心病(CHD)死亡或需要住院治疗的不稳定型心绞痛，总体发生风险降低15％。基于此类临床研究结果，两个抗PCSK9抗体分别于2017年及2019年获得FDA批准用于预防心血管疾病成年患者的心脏病、中风。
发明内容
本申请的一个方面提供PCSK9与LDLR之间的相互作用的抑制剂，其优选为抗PCSK9的抗体或其抗原结合片段，用于治疗高胆固醇血症(更优选纯合型家族性高胆固醇血症，也表示为“HoFH”，或者杂合型家族性高胆固醇血症，也表示为“HeFH”，或者具有心血管极高危或高危风险患者的高胆固醇血症)。
在一些具体实施方案中，所述抗体或其抗原结合片段是单克隆抗体或其抗原结合片段，其特异地结合人PCSK9且阻断人PCSK9与LDLR的结合。
在一些实施方案中，所述抗PCSK9抗体为人源化抗体。
在一些实施方案中，所述抗PCSK9抗体包含：
重链可变区，所述重链可变区含有SEQ ID NO：2所示的氨基酸序列或者与其具有至少80％同源性、优选至少85％同源性、更优选至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同源性的氨基酸序列；以及
轻链可变区，所述轻链可变区含有SEQ ID NO：4所示的氨基酸序列或者与其具有至少80％同源性、优选至少85％同源性、更优选至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同源性的氨基酸序列。
在一些实施方案中，所述抗体的CDR序列如下：
HCDR1：GFTFSSYS(SEQ ID NO：5)；
HCDR2：ISSSSSYI(SEQ ID NO：6)；
HCDR3：EYDFWSAYYDAFDV(SEQ ID NO：7)；
LCDR1：SRNIGGGND(SEQ ID NO：8)；
LCDR2：GVI(SEQ ID NO：9)；以及
LCDR3：QSFDGSLSGSV(SEQ ID NO：10)。
本申请的另一方面提供一种药物组合物，优选用于治疗高胆固醇血症(更优选HoFH、HeFH或具有心血管极高危或高危风险患者的高胆固醇血症)，其包含PCSK9与LDLR之间的相互作用的抑制剂(和他汀类)和药学上可接受的载体。
在一些实施方案中，所述PCSK9与LDLR之间的相互作用的抑制剂为适于胃肠外施用途径或非胃肠外施用途径的形式。
在本申请的另一个方面，提供试剂盒，其优选用于治疗高胆固醇血症(更优选HoFH，HeFH，或者具有心血管极高危或高危风险患者的高胆固醇血症)，其包含本发明所述的抗PCSK9抗体或其抗原结合片段。在一些实施方案中，所述试剂盒还包含他汀类药物，和/或依折麦布。
本申请的目的至少还在于提供一种治疗高胆固醇血症(更优选HoFH，HeFH，或者具有心血管极高危或高危风险患者的高胆固醇血症)的方法，包括向有需要的受试者施用(例如胃肠外或非胃肠外施用)治疗有效量的本发明所述的PCSK9与LDLR之间的相互作用的抑制剂(优选为抗PCSK9的抗体或其抗原结合片段)。优选地，所述受试者被联合施用他汀类药物。更优选地，所述受试者被联合施用他汀类药物和依折麦布。在一些实施方案中，所述抑制剂、他汀类药物和/或依折麦布被同时施用或分开施用。
在一些具体实施方案中，所述他汀类药物为3-羟基-3甲基戊二酰辅酶A(HMG-CoA)还原酶抑制剂。
在一些具体实施方案中，所述他汀类药物，即3-羟基-3甲基戊二酰辅酶A(HMG-CoA)还原酶抑制剂，包括洛伐他汀、普伐他汀、辛伐他汀、氟伐他汀、阿托伐他汀、瑞舒伐他汀和匹伐他汀，或其任意组合等。
在某些实施方案中，所述的高胆固醇血症(更优选HoFH，HeFH，或者具有心血管极高危或高危风险患者的高胆固醇血症)是由LDLR、载脂蛋白B(ApoB)、PCSK9或低密度脂蛋白受体衔接蛋白1(Low Density Lipoprotein Receptor Adaptor Protein 1(LDLRAP1))等的基因中的1个(对)或者更多个(对)等位或非等位基因突变所导致的；其中，所述等位基因突变包括LDLR功能缺陷型突变、影响LDL与LDLR的结合的ApoB基因突变、导致PCSK9对LDLR亲和力增强的功能性突变以及导致LDL内化活性降低的LDLRAP1失功能型突变等。LDLR、APOB或PCSK9基因中已知导致FH的变体参见例如Canadian Journal of Candiology，34，(2018)，1553-1563，Canadian Cardiovascular Society Position Statement on Familial Hypercholesterolemia：Update 2018。
在某些实施方案中，所述的高胆固醇血症(更优选HoFH，HeFH，或者具有心血管极高危或高危风险患者的高胆固醇血症)在既往未经治疗时LDL-C＞13mmol/L(500mg/dL)或(使用大剂量/最大耐受剂量的他汀类药物联合依折麦布)治疗后LDL-C≥8mmol/L(300mg/dL)，同时，合并10岁前出现皮肤或肌腱黄色瘤或父母双亲均携带有LDLR、ApoB、PCSK9或LDLRAP1等基因中的1个(对)或者更多个(对)等位基因突变。
在某些实施方案中，所述的高胆固醇血症(更优选HeFH)满足以下a)和b)：a)所述的高胆固醇血症是如上所述由LDLR、载脂蛋白B(ApoB)、PCSK9或低密度脂蛋白受体衔接蛋白1(Low Density Lipoprotein Receptor Adaptor Protein 1(LDLRAP1))等的基因中的1个(对)或者更多(对)非成对等位基因突变所导致的，b)在既往未经治疗时未接受调脂药物治疗的患者血清LDL-C水平≥4.7mmol/L(180mg/dL)，或者同时满足下述c)和d)的至少1条：c)有皮肤/腱黄色瘤或＜45岁的人存在脂性角膜弓，和d)一级亲属中有FH或早发成人动脉粥样硬化性心血管疾病(ASCVD)，特别是冠心病患者。
本申请的目的至少还在于提供一种本发明所述的PCSK9与LDLR之间的相互作用的抑制剂(优选为抗PCSK9的抗体或其抗原结合片段)在制备治疗高胆固醇血症(更优选HoFH，HeFH，或者具有心血管极高危或高危风险患者的高胆固醇血症)的药物中的用途。
本申请也提供了一种治疗患有高胆固醇血症(更优选HoFH，HeFH，或者具有心血管极高危或高危风险患者的高胆固醇血症)的患者的方法，所述方法包括：(i)对所述患者进行基因型检测，(ii)测量所述患者的样品中的LDL-C水平，和(iii)当所述患者被鉴定为纯合型(或杂合型)家族性高胆固醇血症时，给所述患者施用治疗有效量的本发明所述的抗PCSK9抗体或其抗原结合部分，优选联合施用他汀类药物，更优选联合施用他汀类药物和依折麦布。
在一些实施方案中，所述样品为血清，血浆或全血。
在本发明的某些实施方案中，所述的具有心血管极高危或高危风险患者的高胆固醇血症是指同时符合以下条件1和条件2，其中条件1为符合 a)-h)中的任一条(参考《中国成人血脂异常防治指南(2016年修订版)》)：a)冠状动脉疾病史(如急性冠状动脉综合征、稳定性冠心病、血运重建术后、缺血性心肌病等)，b)缺血性卒中史(腔梗除外)，c)短暂性脑缺血发作，d)外周动脉粥样硬化病(如颈动脉粥样硬化病、颈动脉内膜切除术后、颈动脉支架置入术后、血运重建术后、下肢动脉粥样硬化病、下肢动脉血运重建术后等)，e)LDL-C≥4.9mmol/L(190mg/dl)或TC≥7.2mmol/L，f)1.8mmol/L(70mg/dl)≤LDL-C＜4.9mmol/L(190mg/dl)且年龄在40岁及以上的2型糖尿病患者，g)患有慢性肾脏疾病，分期为3期(即经检测计算的肾小球滤过率30mL/min/1.73m 2≤eGFR＜60mL/min/1.73m 2)，h)存在下列至少3项危险因素：男性≥45岁，女性≥55岁；高血压；吸烟；有CVD家族史(一级亲属：男性＜55岁，女性＜65岁)；HDL胆固醇＜40mg/dL；肥胖(BMI≥28kg/m 2)；并且，同时满足条件2中的a)或b)中的任意一条：存在稳定4周基础降脂药物治疗后患者的血脂水平满足以下条件之一：a)极高危患者LDL-C水平＞1.8mmol/L(70mg/dL)或b)高危患者LDL-C水平＞2.6mmol/L(100mg/dL)的患者。
本申请的目的至少还在于提供一种制备用于治疗高胆固醇血症(优选HoFH，HeFH，或者具有心血管极高危或高危风险患者的高胆固醇血症)的PCSK9与LDLR之间的相互作用的抑制剂(优选抗PCSK9抗体或其抗原结合片段)的药物制剂(药物组合物)的方法。
在一些实施方案中，抗PCSK9抗体的施用治疗以2周(14天)或4周(28天)为一个周期，优选在每个周期第一天(D1)皮下给予抗PCSK9抗体。即，所述抗PCSK9抗体以每两周一次(q2w)或者每四周一次(q4w)的频率施用。
在一些实施方案中，涉及单次药物剂量单元，其包含0.1-60mg按照本发明所述的抗PCSK9抗体或其抗原结合片段，优选用于治疗家族性高胆固醇血症(更优选HoFH，HeFH，或者具有心血管极高危或高危风险患者的高胆固醇血症)。
在本发明的另一个方面，涉及0.1-60mg按照本发明所述的抗PCSK9抗体或其抗原结合片段在制备用于治疗家族性高胆固醇血症(更优选 HoFH，HeFH，或者具有心血管极高危或高危风险患者的高胆固醇血症)的单次药物剂量单元中的应用。
MAB1
如本申请所用，MAB1是抗PCSK9单克隆抗体，其序列和结构可以参见文献(WO 2016/127912 A1)。在所述MAB1单克隆抗体中，HCDR1包含序列GFTFSSYS(SEQ ID NO：5)，HCDR2包含序列ISSSSSYI(SEQ ID NO：6)，HCDR3包含序列EYDFWSAYYDAFDV(SEQ ID NO：7)，LCDR1包含序列SRNIGGGND(SEQ ID NO：8)，LCDR2包含序列GVI(SEQ ID NO：9)，LCDR3包含序列QSFDGSLSGSV(SEQ ID NO：10)。
定义和说明
除非另有说明，本申请中所用的下列术语具有下列含义。一个特定的术语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照本领域普通的含义去理解。当本申请中出现商品名时，意在指代其对应的商品、组合物或其活性成分。
如本文所用，术语“抗体”是指具有至少一个抗原结合结构域的抗原结合蛋白。本申请的抗体和其片段可以是整个抗体或其任何片段。因此，本申请的抗体和其片段包括单克隆抗体或其片段和抗体变体或其片段，以及免疫缀合物。抗体和其片段还可以包括重组多肽、融合蛋白和双特异性抗体。本文公开的抗PCSK9抗体和其片段可以是IgG1、IgG2、IgG3或IgG4同种型。
术语“同种型”是指由重链恒定区基因编码的抗体种类。在一个实施方案中，本文公开的抗PCSK9抗体和其片段是IgG1或IgG4同种型。本申请的抗PCSK9抗体和其片段可以衍生自任何物种，其包括但不限于小鼠、大鼠、兔、灵长类动物、美洲驼和人。PCSK9抗体和其片段可以是嵌合抗体、人源化抗体或完整的人抗体。
术语“人源化抗体”是指其中抗原结合位点来源于非人物种且可变区框架来源于人免疫球蛋白序列的抗体。人源化抗体在框架区中可包含置换，使得该框架可能不是所表达的人免疫球蛋白或种系基因序列的精确拷贝。
术语“基因突变”是指基因组DNA分子发生的突然的、可遗传的变异 现象。从分子水平上看，基因突变是指基因在结构上发生碱基对组成或排列顺序的改变。其包括碱基置换突变、移码突变、缺失突变和插入突变。
“分离的抗体”表示这样的抗体：其基本上不含有具有不同抗原特异性的其它抗体(例如，分离的特异性地结合PCSK9的抗体基本上不含有特异性地结合除PCSK9以外的抗原的抗体)。但是，分离的特异性地结合PCSK9的抗体可以具有与其它抗原(诸如来自不同物种的PCSK9分子)的交叉反应性。此外，分离的抗体可以基本上不含有其它细胞材料和/或化学物质。
术语“单克隆抗体”(“mAb”)是指单分子组合物的抗体分子。单克隆抗体组合物显示出对于特定表位的单一结合特异性和亲和力，或就双特异性单克隆抗体而言，显示出对于两种不同表位的双重结合特异性。mAb是分离的抗体的一个例子。通过本领域技术人员已知的杂交瘤技术、重组技术、转基因技术或其它技术，可以生产mAb。
抗体的“抗原结合部分”(也称为“抗原结合片段”)表示抗体的一个或多个片段，其保留特异性地结合被完整抗体结合的抗原的能力。抗原结合片段的实例包括Fab片段、Fab′片段、F(ab)′片段、Fv片段、分离的CDR区、单链Fv分子(scFv)，F(ab′) 2、Fd、dAb、Fab/c、双价抗体和结构域抗体。
如本文所用，术语“衍生的”当用于指相对于参考抗体或其它结合蛋白的分子或多肽时，意指能够与参考抗体或其它结合蛋白特异性地结合相同表位的分子或多肽。
如本文所用，术语“EC50”是指有效浓度，抗体的50％最大应答。如本文所用，术语“IC50”是指抑制浓度，抗体的50％最大应答。EC50和IC50两者均可以通过ELISA或FACS分析或本领域已知的任何其它方法进行测量。
术语“治疗”一般是指获得需要的药理和/或生理效应的操作。该效应根据完全或部分地预防疾病或其症状，可以是预防性的；和/或根据部分或完全稳定或治愈疾病和/或由于疾病产生的副作用，可以是治疗性的。本文使用的“治疗”涵盖了对患者疾病的任何治疗，包括：(a)预防易感染疾病或症状但还没诊断出患病的患者所发生的疾病或症状；(b)抑制疾病 的症状，即阻止其发展；或(c)缓解疾病的症状，即，导致疾病或症状退化。
如本文所用，术语“全身治疗”是指药物物质通过血流传送，到达并影响全身细胞的治疗。
如本文所用，术语“受试者”表示哺乳动物，诸如啮齿动物、猫科动物、犬科动物和灵长类动物。优选地，根据本申请的受试者是人。
“施用”表示使用本领域技术人员已知的多种方法和递送系统中的任一种，向受试者物理引入包含治疗剂的组合物。免疫检查点抑制剂(例如，抗PCSK9抗体)的施用途径包括静脉内、肌肉内、皮下、腹膜内、脊柱或其它胃肠外施用途径，例如通过注射或输注。本文中使用的短语“胃肠外施用”是指，通常通过注射进行的除了肠内和局部施用以外的施用模式，且包括但不限于，静脉内、肌肉内、动脉内、鞘内、淋巴管内、病灶内、囊内、眶内、心内、真皮内、腹膜内、经气管、皮下、表皮下、关节内、囊下、蛛网膜下、脊柱内、硬膜外和胸骨内注射和输注、以及体内电穿孔。在某些实施方案中，所述免疫检查点抑制剂(例如，抗PCSK9抗体)通过非胃肠外途径施用，在某些实施方案中，口服施用。其它非胃肠外途径包括局部、表皮或粘膜施用途径，例如，鼻内地、阴道地、直肠地、舌下地或局部地。还可以执行施用，例如，一次、多次，和/或在一个或多个延长的时间段中。
本文中使用的“不良事件”(AE)是与医学治疗的应用有关的任何不利的和通常非故意的或不希望的迹象(包括异常的实验室发现)、征状或疾病。例如，不利事件可以与响应于治疗的分泌PCSK9的肝脏相关。医学治疗可以具有一种或多种相关的AE，并且每种AE可以具有相同或不同的严重性水平。对能够“改变不利事件”的方法的提及是指降低与不同治疗方案的应用相关的一种或多种AE的发生率和/或严重性的治疗方案。
本文中使用的“给药间隔”是指在施用给受试者的本文公开的制剂的多个剂量之间逝去的时间的量。因而可以将给药间隔指示为范围。
本文中使用的术语“给药频率”表示在给定时间中本文公开的制剂的施用剂量的频率。可以将给药频率指示为每个给定时间的给药次数，例如，每周1次或2周1次。
术语“统一剂量(flat dose)”的应用是指，不考虑患者的重量或体表面积(BSA)施用给患者的剂量。因此将统一剂量规定为药剂(例如，抗PCSK9抗体)的绝对量，而不是规定为mg/kg剂量。例如，60kg人和100kg人将接受相同剂量的抗体(例如，240mg抗PCSK9抗体)。
关于本申请的组合物的术语“固定剂量”的应用是指，单一组合物中的两种或更多种不同抗体彼此以特定(固定)比率存在于所述组合物中。在某些实施方案中，所述固定剂量是基于所述抗体的重量(例如，mg)。在某些实施方案中，所述固定剂量是基于所述抗体的浓度(例如，mg/ml)。在某些实施方案中，所述第一抗体(mg)：第二抗体(mg)的比率是至少约1∶1、约1∶2、约1∶3、约1∶4、约1∶5、约1∶6、约1∶7、约1∶8、约1∶9、约1∶10、约1∶15、约1∶20、约1∶30、约1∶40、约1∶50、约1∶60、约1∶70、约1∶80、约1∶90、约1∶100、约1∶120、约1∶140、约1∶160、约1∶180、约1∶200、约200∶1、约180∶1、约160∶1、约140∶1、约120∶1、约100∶1、约90∶1、约80∶1、约70∶1、约60∶1、约50∶1、约40∶1、约30∶1、约20∶1、约15∶1、约10∶1、约9∶1、约8∶1、约7∶1、约6∶1、约5∶1、约4∶1、约3∶1或约2∶1。例如，第一抗体和第二抗体的3∶1比率可以是指，瓶中可以含有约240mg第一抗体和80mg第二抗体，或约3mg/ml的第一抗体和1mg/ml的第二抗体。
“患者”(有时表示为“受试者”)包括任何人或非人动物。术语“非人动物”包括但不限于脊椎动物诸如非人灵长类动物、绵羊、狗，和啮齿类动物诸如小鼠、大鼠和豚鼠。在某些实施方案中，所述患者是人。术语“受试者”和“患者”在本文中的某些语境下可互换地使用。
药物或治疗剂的“治疗有效量”或“治疗上有效的剂量”是当单独使用或与另一种治疗剂联合使用时保护患者免于疾病发作或促进疾病消退的药物的任何量，所述疾病消退通过疾病征状的严重程度的降低、无疾病征状阶段的频率和持续时间的增加、或由疾病折磨引起的损伤或失能的预防来证明。使用熟练的从业人员已知的多种方法可以评价治疗剂的促进疾病消退的能力，诸如在临床试验期间在人患者中，在预测对于人类的效力的动物模型系统中，或通过在体外测定法中测定所述药剂的活性。
术语“约”、“大约”或“基本上包含”表示在本领域普通技术人员确定的 特定值或组成的可接受误差范围内的值或组成，其将部分地取决于如何测量或测定值或组成，即，测量系统的限制。例如，“约”、“大约”或“基本上包含”可以是指按本领域中的实践，在1个或超过1个标准差内。可替换地，“约”或“基本上包含”可以是指与由其修饰的参数或数值相差至多10％或20％(即，±10％或±20％)的范围。例如，约3mg可以包括2.7mg至3.3mg之间(对于10％)或2.4mg至3.6mg(对于20％)之间的任何数字。此外，特别是关于生物学系统或过程，该术语可以是指直到一个数量级或直到数值的至多5倍。当在本申请和权利要求中提供特定值或组成时，除非另有说明，否则“约”或“基本上包含”的含义应当假定在该特定值或组成的可接受误差范围内。
本文中使用的术语“约每周一次”、“约每两周一次”或任意其它类似的给药间隔术语是指近似值。“约每周一次”可以包括每7天±1天，即，每6天至每8天。“约每两周一次”可以包括每14天±3天，即，每11天至每17天。类似的近似值适用于，例如，约每3周一次，约每4周一次，约每5周一次，约每6周一次，和约每12周一次。在某些实施方案中，约每6周一次或约每12周一次的给药间隔是指可以在第一周的任意天施用第一剂，然后可以分别在第六周或第十二周的任意天施用第二剂。在其它实施方案中，约每6周一次或约每12周一次的给药间隔是指在第一周的特定天(例如，星期一)施用第一剂，并然后分别在第六周或第十二周的相同天(即，星期一)施用第二剂。类似的原则适用于包括、但不限于“约每2周1次”，“约每月1次”等......的短语。
如本文中所述的，任何浓度范围、百分比范围、比率范围或整数范围应当理解为包括在列举的范围内的任意整数的值，且当适当时，包括其分数(诸如整数的十分之一和百分之一)，除非另外指出。
除特别声明外，本申请中的“约”或“大约”是指在所给定的具体数值范围±5％范围内波动，优选在±2％范围内波动，更优选在±1％范围内波动。例如pH值为约5.5表示pH为5.5±5％，优选pH为5.5±2％，更优选pH为5.5±1％。
术语“药学上可接受的”是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接 触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。
术语“药物的制剂”、“药物组合物”是指一种本申请的活性成分(例如抗PCSK9抗体)与药学上可接受的辅料组成的混合物。药物组合物的目的是有利于对受试者给予本申请的活性成分。
术语“单次药物剂量单元”表示在给药方案时刻，(优选按照受试者每kg体重)待给药于受试者的包含本发明所述的抗PCSK9抗体或其抗原结合片段的单次药物剂型，如安瓿瓶。
施用方式
下述内容并非限制本申请药物的施用方式。
在一个实施方案中，本申请的药物可以配制成适合于单次或多次施用的药物组合物。
本申请的药物以适合的各种途径施用，包括但不限于，口服或肠胃外(通过静脉内、肌内、局部或皮下途径)。在一些实施方案中，本申请的药物口服施用或注射施用，例如静脉注射或腹腔注射。
本申请的药物适合的剂型，包括但不限于，片剂、含片、丸剂、胶囊剂(例如硬胶囊、软胶囊、肠溶胶囊、微囊剂)、酏剂、颗粒剂、糖浆剂、注射剂(肌肉内、静脉内、腹腔内)、颗粒剂、乳剂、悬浮液、溶液、分散剂和用于口服或非口服给药的缓释制剂的剂型。
本申请的药物组合物含有药学上可接受的载体和/或赋形剂。
附图说明
图1.MAB1对小鼠血清LDL-C的影响。
图2.MAB1对食蟹猴血清LDL-C的影响，s.c：皮下注射；i.v：静脉注射；male：雄性；；female：雌性。
图3.MAB1对HepG2细胞的LDL的内吞作用的影响。
图4.MAB1解除野生型PCSK9对LDL内吞的抑制。
图5.MAB1解除功能增强突变型PCSK9(PCSK9-D374Y)对LDL内吞的抑制。
具体实施方式
下面结合具体实施例对本申请进行进一步的描述，然而，本申请中这些实施例仅用于阐明而不限制本申请的范围。同样，本申请不限于本文描述的任何具体优选的实施方案。本领域技术人员应该理解，对本申请技术特征所作的等同替换、或相应的改进仍属于本申请的保护范围之内。除特别说明的以外，以下实施例采用的试剂均为市售产品，溶液的配制可以采用本领域常规技术。
在本发明的下述实施例中，同型对照抗体为人抗鸡蛋溶酶体(human anti-Hen Egg Lysozyme IgG，anti-HEL，即human IgG，简称hIgG)，其序列来自于Acierno等人发表的Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies(Acierno等人.J Mol Biol.2007；374(1)：130-46.，其中重链的氨基酸序列为SEQ ID NO：11所示，轻链的氨基酸序列为SEQ ID NO：12所示)，设计为本研究的同型对照抗体，在中山康方生物医药有限公司实验室制备而成。
在本发明的下述实施例中，使用的C57BL/6小鼠购自广东省医学实验动物中心。
实施例1.人源化抗PCSK9抗体的制备
1.抗体的设计
为制备单克隆抗体MAB1，本发明人根据已有的PCSK9蛋白序列(NP_777596.2)及其蛋白三维晶体结构等，人工设计了一系列的抗体序列。通过大量的筛选和检测，最终得到了一种与PCSK9特异性结合的MAB1。该单克隆抗体重链和轻链可变区的氨基酸序列及其编码序列如下面的SEQ ID NO：1-4。
设计的MAB1重链VH的DNA序列如下(369bp)：
Figure PCTCN2020129763-appb-000001
Figure PCTCN2020129763-appb-000002
其编码的VH蛋白序列如下(123aa)：
Figure PCTCN2020129763-appb-000003
MAB1轻链VL的DNA序列如下(333bp)：
Figure PCTCN2020129763-appb-000004
其编码的VL蛋白序列如下(111aa)：
Figure PCTCN2020129763-appb-000005
所述抗体的CDR序列如下：
HCDR1：GFTFSSYS(SEQ ID NO：5)；
HCDR2：ISSSSSYI(SEQ ID NO：6)；
HCDR3：EYDFWSAYYDAFDV(SEQ ID NO：7)；
LCDR1：SRNIGGGND(SEQ ID NO：8)；
LCDR2：GVI(SEQ ID NO：9)；以及
LCDR3：QSFDGSLSGSV(SEQ ID NO：10)。
2.抗体的表达和纯化
将MAB1的重链cDNA序列(编码序列如SEQ ID NO：1所示；恒定区是Ig gamma-1 chain C region，ACCESSION：P01857)和轻链的cDNA序列(编码序列如SEQ ID NO：3所示；恒定区为Ig lambda-2 chain C regions；ACCESSION：P0CG05.1)分别克隆到pUC57simple(金斯瑞公司提供)载体中，分别获得pUC57simple-MAB1H和pUC57simple-MAB1L质粒。分别将质粒pUC57simple-MAB1H和pUC57simple-MAB1L进行酶切(HindIII&EcoRI)，电泳回收得到的重链轻链分别亚克隆到pcDNA3.1载体中，提取重组质粒共转染293F细胞。细胞培养7天后，将培养液通过高速离心、上清浓缩后上样至HiTrap MabSelect SuRe柱，用Elution Buffer一步洗脱蛋白并回收目标样品并换液至PBS。
实施例2.MAB1对动物血清低密度脂蛋白胆固醇LDL-C的影响
(1) MAB1对小鼠血清低密度脂蛋白胆固醇LDL-C的影响
32只C57BL/6小鼠给药前通过眼眶采血分离血清，使用全自动生化分析仪(迈瑞，型号BS-180)检测小鼠低密度脂蛋白胆固醇(LDL-C)。如表1所示，将小鼠分为4组，即正常组、模型组、MAB1低剂量组和MAB1高剂量组，每组8只，并按表1给药方案给药。并在给药前D0、给药后D3、D7、D10、D14、D18采血分离血清，进行血脂低密度脂蛋白胆固醇检测。
实验结果如图1所示。实验结论，与模型组相比，MAB1低剂量60mg/kg组和MAB1高剂量120mg/kg组均能够明显降低小鼠血清中LDL-C含量(P＜0.05或P＜0.01)。
表1：MAB1对小鼠的给药剂量和给药方案
Figure PCTCN2020129763-appb-000006
Figure PCTCN2020129763-appb-000007
注：第一次给药时间为D0，即第0天；皮下：即皮下注射；
(2) MAB1对食蟹猴血清低密度脂蛋白胆固醇LDL-C的影响
选用从南宁市德恒生物科技有限公司(动物生产许可证：SCXK(桂)2011-0007，实验动物合格证编号：0005869)购买的12只雄性和12只雌性食蟹猴(普通级)进行本实验。所有涉及动物使用和福利的操作和活动都要在操作前经机构动物伦理委员会(IACUC)批准。IACUC批准编号为：ACU16-467。
如表2所示，根据给药前测定的动物体重，使用计算机系统将动物按性别随机分配到MAB1低剂量组、MAB1中剂量组、MAB1高剂量组和MAB1静脉给药组，并按表2给药方案给药。并在所有动物于药前D0、药后4h，D2、D4、D6、D8、D11、D15、D18、D22，空腹采集血样约0.5ml。血样采集后转移至离心管中，于2-8℃、3000g条件下离心10分钟。测定低密度脂蛋白(LDL-C)。
实验结果，如图2所示。实验结论，MAB1以3、10及30mg/kg剂量单次皮下注射给予食蟹猴，以10mg/kg单次静脉注射给予食蟹猴。食蟹猴体内的低密度脂蛋白，在给药后均显著下降。至药后约15天，3mg/kg剂量组动物血脂水平基本回复至药前水平，至药后22天，10mg/kg的皮下注射及静脉注射剂量组动物血脂水平也基本回复，而30mg/kg剂量组部分动物血脂水平仍明显低于药前。
表2：MAB1对食蟹猴的给药剂量和给药方案
Figure PCTCN2020129763-appb-000008
Figure PCTCN2020129763-appb-000009
注：第一次给药时间为D0，即第0天；皮下：即皮下注射；静脉；即静脉注射；根据给药前测定的动物体重，使用计算机系统将动物按性别随机分配到4组中。每组雌性3只，雄性3只；
实施例3.MAB1对HepG2细胞的LDL的内吞作用的影响
PCSK9可以通过介导LDLR的内吞分解进而抑制HepG2细胞表面LDLR的表达。LDL可以结合LDLR进而被内吞入细胞，BODIPY荧光标记LDL(BODIPY-LDL，Life，货号：L3483)可作为标记物与LDLR结合并被内吞入细胞，通过检测细胞内BODIPY-LDL的荧光值，进而反映LDLR介导的LDL的内吞。如PCSK9存在并结合于LDLR，则会导致细胞表面的LDLR减少进而结合于LDLR的BODIPY-LDL减少，即抑制了LDL的内吞；反之，如抑制PCSK9则可以增强HepG2细胞对LDL内吞。因此，该实验体系可以通过检测HepG2细胞对BODIPY-LDL的内吞活性而衡量抗PCSK9抗体的药理学活性。
实验方法：每孔加入50μl多聚赖氨酸(Poly-l-lysine)溶液包被96孔黑底细胞培养板，置于37℃，5％CO 2培养箱中2小时，弃包被液，100μl PBS洗涤一次，晾干待用。HepG2细胞常规消化，以分析培养基(1％NEAA的DMEM)重悬细胞，计数2×10 4/孔接种于96孔黑底细胞培养板中，每孔加入80μl，置于37℃，5％CO 2培养箱中培养18小时。用分析培养基(1％NEAA的DMEM)预稀释PCSK9至6000nM，每孔加入10μl；抗体稀释成10×，每孔加入10μl，置于37℃，5％CO 2培养箱中培养6小时。之后，每孔加入10μl 0.1mg/ml的BODIPY-LDL溶液，置于37℃，5％CO 2培养箱中培养22小时。取出培养板，弃去培养基，每孔加入200μl PBS洗涤后弃去。加入甲醛溶液以固定细胞，室温放置20min。弃去甲醛，加入PBS洗涤。最后弃去PBS，每孔加入100μl PBS用多标记微孔板检测 仪设置激发光波长490nm，发射波长520nm，cutoff为515nm读取BODIPY的相对荧光单位数值。
结果如表3和图3所示，MAB1解除PCSK9抑制HepG2细胞内吞LDL活性的EC50值为12.99μg/ml。
实验结论，MAB1能抑制PCSK9的活性，从而使HepG2细胞内吞LDL的量增加，MAB1的EC50为12.99μg/ml。
表3 MAB1检测HepG2细胞的LDL的内吞作用结果
Figure PCTCN2020129763-appb-000010
实施例4.MAB1解除野生型和功能增强突变型PCSK9对的LDL内吞的抑制
PCSK9存在多种突变形式，根据突变对PCSK9调节LDL-C水平影响不同分为两类，功能获得型和功能缺失型。功能获得型突变可增强PCSK9上调LDL-C的能力，导致高胆固醇血症，主要形式有S127R、D129G、F216L、R218S、D374Y、N425S、R496W、H553R和E670G等。
其中，D374Y(Asp374Tyr)突变改变PCSK9、LDLR亲和力，使得PCSK9、LDLR亲和力增加5至30倍(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)。
本实验以D374Y(Asp374Tyr)突变蛋白为例，研究了MAB1对于抑制功能增强型PCSK9突变蛋白介导的LDLR的内吞的活性。实验结果提 示，MAB1可有效解除功能获得型PCSK9蛋白对LDL的内吞的抑制。
实验材料：HepG2(购自中国科学院，P14)；DMEM(Gibco，货号：11995040)；FBS(Excell bio，货号：FSP500)；MEM NEAA(100X)(Gibco，货号：11140-050)；POLY-L-LYSINE(SIGMA，货号：P4832)；BODIPY-LDL(Life，货号：L3483)；PCSK9-his(康方生物自产)；PCSK9(D374Y)-his(Acrobio，货号：PCY-H5225)。Evolocumab(购自AMGEN)。
实验方法：50μL 0.01％的多聚赖氨酸包被黑色96孔细胞培养板，置于37℃，5％CO 2培养箱2h，弃包被液，100μL PBS洗涤一次，晾干待用。处于对数期的HepG2细胞，弃去培养皿中的生长培养基(12％FBS，1％NEAA的DMEM)，胰蛋白酶消化后以分析培养基(1％NEAA的DMEM)重悬细胞，计数后，20000/well接种于黑色的96孔细胞培养板中，每孔80μL，37℃，5％CO 2条件下培养过夜。次日，以分析培养基稀释PCSK9和抗体(终浓度如图所示)，加入到96孔板中，体系终体积为100μL。将细胞培养板放置到37℃，5％CO 2条件下培养21h。取出培养板，每孔加入10μL 0.1mg/mL BODIPY标记的LDL溶液，将培养板置于37℃，5％CO 2条件下培养9h。从培养箱取出培养板，弃去培养基，每孔加入200μL PBS洗涤培养板后弃去。每孔加入100μL PBS用多标记微孔板检测仪，以485nm为激发光波长，以535nm为发射光波长，在细胞培养板上读取相对荧光单位(Relative Fluorescence units，RFU)数值。
实验结果：实验结果如表4，图4，图5所示，MAB1可有效的与野生型PCSK9及功能增强突变型PCSK9(PCSK9-D374Y)结合，有效阻断PCSK9对LDL内吞的抑制，其与同靶点已上市药物Evolocumab活性相当。
表4.MAB1抑制野生型和功能增强突变型PCSK9对LDLR内吞的EC50
Figure PCTCN2020129763-appb-000011
实施例4.MAB1解除野生型PCSK9蛋白对突变型LDLR介导的LDL内吞的抑制
PCSK9蛋白结合于LDLR发挥调控LDL-C代谢的功能。LDLR基因突变的众多，以LDLR蛋白合成与功能为基础突变可划分为1)LDLR不表达型突变，包括LDLR等位基因不表达即细胞不表达LDLR，具体的有启动子序列突变、无义突变、移码突变、剪接突变等，以及LDLR转运缺陷即有缺陷的LDLR合成后无法从内质网向高尔基体的转运进而堆积胞内被分解；2)为结合缺陷型基因突变，即突变基因编码的异常LDLR可转运并在细胞表面表达，但是结合LDL-C的活性下降或者消失；3)内移缺陷型基因突变，即由于编码NPVY序列的密码子发生突变，受体能与LDL结合但不能被内吞从而聚集于被覆陷窝；4)再循环缺陷型基因突变，即受体能够结合LDL-C并介导内吞但是将会在溶酶体内与LDL-C同时降解，导致LDLR不能够再循环到细胞表面。本实验以野生型LDLR(即对应下面突变型LDLR(D227E)的第227位氨基酸为D)，突变型LDLR(D227E)(https：//www.ncbi.nlm.nih.gov/clinvar/variation/3690/)作为对照，评价不同突变型LDLR内吞LDL活性、PCSK9对不同突变型LDLR内吞LDL的抑制以及MAB1是否有效解除PCSK9介导的LDL内吞的抑制作用。
实验方法：293T细胞常规消化铺板，5*10 5/孔接种至6孔板中，2mL DMEM+10％FBS+1％双抗(Penicillin Streptomycin，Gibco，货号：15140-122)，置于培养箱孵育过夜；转染前2h换液，换成1mL的Opti-MEM无血清培养基；Opti-MEM分别稀释目的质粒DNA(中山康方生物医药有限公司构建，见表5，以野生型LDLR为基础，以起始密码子编码的氨基酸为位置1，按照表5进行相应位置的定点突变后，插入到购自YouBio公司的慢病毒质粒pCDH-CMV-MCS-EF1-puro，获得表5中的各个质粒)和PEI(Polyscience，货号：23966)，目的质粒与对照质粒(中山康方生物医药有限公司构建，见表5)(各2μg，PEI 10μg(单转的PEI用量5μg)，稀释好的DNA和PEI混匀孵育15min后缓慢滴加到6孔板内，轻轻摇匀，置于培养箱孵育过夜；将6孔板内培养基换成2mL DMEM+10％FBS，置于培养箱继续培养；转染48h后，0.05％胰酶-EDTA 消化离心收集细胞，计数；取部分细胞做reporter assay实验，2*10 5/孔，100μL DMEM+10％FBS，PBS和IL-6(康方生物，批号：20150421)(50ng/mL)组，孵育过夜后加50μL底物(Bright-Glo TMluciferase assay system，Promega，货号：E2620)读取RLU值；取部分细胞做LDL内吞(FACS)实验，200μL Opti-MEM+1％NEAA，2*10 5/孔，PCSK-9、hIgG1(康方生物，批号：20170424)、MAB1终浓度分别为600nM、50μg/mL、50μg/mL，PCSK-9与抗体预孵育后给药，与细胞孵育过夜。
按实验设计加入对应的LDL(BODIPY-LDL，Life，货号：L3483)(终浓度为0.001mg/mL)，混匀孵育4h；
收集细胞转移至尖底96孔板中，350xg离心5min，弃上清；加200μL1％PBSA重悬细胞沉淀，350xg离心5min，弃上清，重复洗涤一次；加200μL 1％PBSA重悬细胞沉淀，转移至流式管中，上机检测。
实验结果如表5，以野生型LDLR为例，LDL内吞率为11.50％，加入PCSK9后内吞率为7.23％，表明PCSK9阻断了LDLR对LDL的内吞，在LDL和PCSK9的基础上加入MAB1，内吞率为8，53％，表明MAB1可有效解除PCSK9介导的LDL内吞的抑制作用。表中突变序列信息均来源于文献及相关在线数据库(Liang L et al.，Scientific Reports.2015；5(1)：17272.； https：//www.ncbi.nlm.nih.gov/clinvar/)。
表5野生型LDLR和不同突变型LDLR对BODIPY-LDL的内吞率％
Figure PCTCN2020129763-appb-000012
研究结果显示，当突变型LDLR具有内吞LDL活性，且PCSK9能抑制其对LDL的内吞时，加入MAB1可显著增加LDL的内吞，即MAB1可有效解除PCSK9介导的LDL内吞的抑制作用。

Claims (18)

  1. 抗PCSK9的抗体或其抗原结合片段，用于治疗高胆固醇血症。
  2. 权利要求1的抗PCSK9的抗体或其抗原结合片段，其中所述抗PCSK9抗体包含：
    HCDR1：GFTFSSYS(SEQ ID NO：5)；
    HCDR2：ISSSSSYI(SEQ ID NO：6)；
    HCDR3：EYDFWSAYYDAFDV(SEQ ID NO：7)；
    LCDR1：SRNIGGGND(SEQ ID NO：8)；
    LCDR2：GVI(SEQ ID NO：9)；以及
    LCDR3：QSFDGSLSGSV(SEQ ID NO：10)。
  3. 权利要求1的抗PCSK9的抗体或其抗原结合片段，其中所述抗PCS9抗体包含重链可变区，所述重链可变区含有SEQ ID NO：2所示的氨基酸序列或者与其具有至少80％同源性、优选至少85％同源性、更优选至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同源性的氨基酸序列；以及
    轻链可变区，所述轻链可变区含有SEQ ID NO：4所示的氨基酸序列或者与其具有至少80％同源性、优选至少85％同源性、更优选至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同源性的氨基酸序列。
  4. 权利要求1的抗PCSK9的抗体或其抗原结合片段，其中所述抗体是单克隆抗体，优选人源化抗体。
  5. 药物组合物，优选用于治疗高胆固醇血症，其包含权利要求1-4任一项所述的抗PCSK9的抗体或其抗原结合片段，和药学上可接受的载体。
  6. 试剂盒，优选用于治疗高胆固醇血症，其包含权利要求1-4任一项所述的抗PCSK9抗体或其抗原结合片段，优选地，所述试剂盒还包含他汀类药物(优选3-羟基-3甲基戊二酰辅酶A(HMG-CoA)还原酶抑制剂，更优选选自洛伐他汀、普伐他汀、辛伐他汀、氟伐他汀、阿托伐他汀、瑞舒伐他汀和匹伐他汀)，和/或依折麦布。
  7. 权利要求6所述的试剂盒，其中所述抗PCSK9抗体为适于胃肠外施用途径或非胃肠外施用途径的形式。
  8. 治疗高胆固醇血症的方法，包括向有需要的受试者施用(例如胃肠外或非胃肠外施用)治疗有效量的权利要求1-4任一项所述的抗PCSK9抗体其抗原结合片段，优选地，所述受试者被联合施用他汀类药物，更优选地，所述受试者被联合施用他汀类药物和依折麦布。
  9. 权利要求8的方法，其中所述抗PCSK9抗体或其抗原结合片段，所述他汀类药物和/或依折麦布被同时施用或分开施用。
  10. 权利要求8的方法，其中所述治疗有效量为根据患者体重，0.1mg/kg以上，优选0.1-60mg/kg。
  11. 权利要求1-4任一项所述的抗PCSK9抗体或其抗原结合片段，权利要求5所述的药物组合物，权利要求6-7任一项所述的试剂盒，或权利要求8-9任一项所述的方法，其中所述高胆固醇血症为选自以下各项组成的组：
    (1)纯合型家族性高胆固醇血症，更优选地，所述高胆固醇血症是由LDLR、载脂蛋白B(ApoB)、PCSK9或低密度脂蛋白受体衔接蛋白1(Low Density Lipoprotein Receptor Adaptor Protein 1(LDLRAP1))的基因中的1个(对)或者更多个(对)等位或非等位基因突变所导致的，优选地，所述等位基因突变包括LDLR功能缺陷型突变、影响LDL与LDLR的结合的ApoB基因突变、导致PCSK9对LDLR亲和力增强的功能性突变以及导致LDL内化活性降低的LDLRAP1失功能型突变，和/或优选地，所述高胆固醇血症(更优选HoFH)在既往未经治疗时LDL-C＞13mmol/L(500mg/dL)或(使用大剂量/最大耐受剂量的他汀类药物联合依折麦布)治疗后LDL-C≥8mmol/L(300mg/dL)，同时，合并10岁前出现皮肤或肌腱黄色瘤或父母双亲均携带有LDLR、ApoB、PCSK9或LDLRAP1基因中的1对或者更多对等位基因突变；
    (ii)杂合型家族性高胆固醇血症，优选地，所述杂合型家族性高胆固醇血症由LDLR、载脂蛋白B(ApoB)、PCSK9或低密度脂蛋白受体衔接蛋白1(Low Density Lipoprotein Receptor Adaptor Protein 1(LDLRAP1))基因中的1个或者更多非成对等位基因突变所导致的，更优 选地，所述等位基因突变包括LDLR功能缺陷型突变、影响LDL与LDLR的结合的ApoB基因突变、导致PCSK9对LDLR亲和力增强的功能性突变以及导致LDL内化活性降低的LDLRAP1失功能型突变；
    (iii)具有心血管极高危或高危风险患者的高胆固醇血症，优选所述具有心血管极高危或高危风险患者的高胆固醇血症是指同时符合以下条件1和条件2，其中条件1为符合a)-h)中的任一条：a)冠状动脉疾病史(如急性冠状动脉综合征、稳定性冠心病、血运重建术后、缺血性心肌病等)，b)缺血性卒中史(腔梗除外)，c)短暂性脑缺血发作，d)外周动脉粥样硬化病(如颈动脉粥样硬化病、颈动脉内膜切除术后、颈动脉支架置入术后、血运重建术后、下肢动脉粥样硬化病、下肢动脉血运重建术后等)，e)LDL-C≥4.9mmol/L(190mg/dl)或TC≥7.2mmol/L，f)1.8mmol/L(70mg/dl)≤LDL-C＜4.9mmol/L(190mg/dl)且年龄在40岁及以上的2型糖尿病患者，g)患有慢性肾脏疾病，分期为3期(即经检测计算的肾小球滤过率30mL/min/1.73m 2≤eGFR＜60mL/min/1.73m 2)，h)存在下列至少3项危险因素：男性≥45岁，女性≥55岁；高血压；吸烟；有CVD家族史(一级亲属：男性＜55岁，女性＜65岁)；HDL胆固醇＜40mg/dL；肥胖(BMI≥28kg/m 2)；并且，同时满足条件2中的a)或b)中的任意一条：存在稳定4周基础降脂药物治疗后患者的血脂水平满足以下条件之一：a)极高危患者LDL-C水平＞1.8mmol/L(70mg/dL)或b)高危患者LDL-C水平＞2.6mmol/L(100mg/dL)的患者。
  12. 治疗患有高胆固醇血症的患者的方法，所述方法包括：(i)对所述患者进行基因型检测，(ii)测量所述患者的样品(优选血清，血浆或全血)中的LDL-C水平，和(iii)当所述患者被鉴定为纯合型或杂合型家族性高胆固醇血症时，给所述患者施用治疗有效量的抗PCSK9抗体或其抗原结合片段，优选联合施用他汀类药物，更优选联合施用他汀类药物和依折麦布，优选地，所述治疗有效量为根据患者体重，0.1mg/kg以上，优选0.1-60mg/kg。
  13. 权利要求12所述的方法，其中纯合型或杂合型家族性高胆固醇血症如权利要求11中所定义。
  14. 权利要求12-13任一项所述的方法，其中所述患者为杂合型家族 性高胆固醇血症患者时，所述患者在既往未经治疗时未接受调脂药物治疗的患者血清LDL-C水平≥4.7mmol/L(180mg/dL)，或者，
    所述患者为杂合型家族性高胆固醇血症患者时，所述患者同时满足下述a)和b)的至少1条：a)有皮肤/腱黄色瘤或＜45岁的人存在脂性角膜弓，和b)一级亲属中有FH或早发成人动脉粥样硬化性心血管疾病(ASCVD)，特别是冠心病患者。
  15. 权利要求8-12任一项所述的方法，其中抗PCSK9抗体或其抗原结合片段的施用治疗以2周(14天)或4周(28天)为一个周期，优选在每个周期第一天(D1)皮下给药。
  16. 权利要求1-4任一项所述的抗PCSK9抗体或其抗原结合片段在制备用于降低具有心血管极高危或高危风险的高胆固醇血症患者心血管疾病的发作及死亡风险的药物制剂(或药物组合物)中的应用。
  17. 降低高血脂心血管疾病患者(更优选极高/高危心血管疾病风险患者)的血胆固醇或降低高血脂患者心血管疾病发作的风险的方法，包括给所述患者施用(例如胃肠外或非胃肠外施用)治疗有效量的权利要求1-4任一项所述的抗PCSK9抗体其抗原结合片段，优选地，所述受试者被联合施用他汀类药物，更优选地，所述受试者被联合施用他汀类药物和依折麦布，更优选地，所述他汀类药物选自洛伐他汀、普伐他汀、辛伐他汀、氟伐他汀、阿托伐他汀、瑞舒伐他汀和匹伐他汀或其任意组合。
  18. 权利要求17所述的方法，抗PCSK9抗体的施用治疗以2周(14天)或4周(28天)为一个周期，优选在每个周期第一天(D1)皮下给予抗PCSK9抗体。
PCT/CN2020/129763 2019-11-18 2020-11-18 抗pcsk9抗体及其应用 Ceased WO2021098720A1 (zh)

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CA3160071A CA3160071A1 (en) 2019-11-18 2020-11-18 Anti-pcsk9 antibody and use thereof
KR1020227020280A KR20220107210A (ko) 2019-11-18 2020-11-18 항-pcsk9 항체 및 그의 용도
IL323016A IL323016A (en) 2019-11-18 2020-11-18 Anti-pcsk9 antibody and its use
EP20888942.8A EP4063396A4 (en) 2019-11-18 2020-11-18 ANTI-PCSK9 ANTIBODIES AND ITS USE
IL292973A IL292973B2 (en) 2019-11-18 2020-11-18 Anti-PCSK9 antibody and its use
BR112022009587A BR112022009587A2 (pt) 2019-11-18 2020-11-18 Anticorpo anti-pcsk9 e uso do mesmo
AU2020388182A AU2020388182A1 (en) 2019-11-18 2020-11-18 Anti-PCSK9 antibody and use thereof
US17/776,295 US20230312750A1 (en) 2019-11-18 2020-11-18 Anti-pcsk9 antibody and use thereof
JP2022528582A JP2023501745A (ja) 2019-11-18 2020-11-18 抗ｐｃｓｋ９抗体及びその使用
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