WO2016127912A1 - Pcsk9抗体、其药物组合物及其用途 - Google Patents

Pcsk9抗体、其药物组合物及其用途 Download PDF

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WO2016127912A1
WO2016127912A1 PCT/CN2016/073492 CN2016073492W WO2016127912A1 WO 2016127912 A1 WO2016127912 A1 WO 2016127912A1 CN 2016073492 W CN2016073492 W CN 2016073492W WO 2016127912 A1 WO2016127912 A1 WO 2016127912A1
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antibody
monoclonal antibody
antigen
pcsk9
binding fragment
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PCT/CN2016/073492
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English (en)
French (fr)
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李百勇
王忠民
夏瑜
张鹏
任武贤
焦进安
徐元元
戴东升
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中山康方生物医药有限公司
苏州亚宝药物研发有限公司
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Application filed by 中山康方生物医药有限公司, 苏州亚宝药物研发有限公司 filed Critical 中山康方生物医药有限公司
Priority to US15/550,211 priority Critical patent/US10670604B2/en
Priority to KR1020177024712A priority patent/KR102531577B1/ko
Priority to JP2017542863A priority patent/JP6731933B2/ja
Publication of WO2016127912A1 publication Critical patent/WO2016127912A1/zh
Priority to US16/855,202 priority patent/US11402383B2/en

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    • A—HUMAN NECESSITIES
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    • A61P3/06—Antihyperlipidemics
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—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
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • 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
    • C07K2317/565—Complementarity determining region [CDR]
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/321—Arterial hypertension

Definitions

  • the invention belongs to the field of immunology and molecular biology, and relates to a PCSK9 antibody, a pharmaceutical composition thereof and use thereof.
  • the invention relates to monoclonal antibodies to PCSK9.
  • PCSK9 is a subtilisin protease having proprotein convertase activity.
  • PCSK9 is expressed in the liver, small intestine and kidney, and is expressed in a small amount in the skin and nervous system.
  • PCSK9 in the liver can be secreted into the blood circulation and reduce the number of cell surface low density lipoprotein receptors (LDLRs) by binding and endocytosis. Because LDLR can efficiently eliminate LDL from plasma, a decrease in the number of LDLR receptors can promote LDL accumulation, so PCSK9-mediated LDLR degradation can increase plasma low-density lipoprotein cholesterol (LDL-C) levels.
  • LDL-C plasma low-density lipoprotein cholesterol
  • PCSK9 is involved in the lipid metabolism of triglyceride-rich apolipoprotein B.
  • the secretion of PCSK9 can increase the level of intestinal triglyceride (TG), which is involved in the induction of hypercholesterolemia (Rashid S., et al., Proprotein convertase Subtilisin kexin type 9 promotes intestinal overproduction of triglyceride-rich apolipoprotein B lipoproteins through both low-density lipoprotein receptor-dependent and-independent mechanisms. Circulation. 2014 Jul 29;130(5):431-41.).
  • statins Twenty-nine percent of patients with hypercholesterolemia use statins. Patients who could not tolerate statins and statins that did not achieve the required levels of cholesterol and cholesterol accounted for 8.2% of patients with hypercholesterolemia.
  • the world's seven major drug markets (excluding China) have 390 million patients with hypercholesterolemia.
  • the “Research on Nutrition and Health Status of Chinese Residents” published by the Ministry of Health showed that the prevalence of hypertension in adults in China was 18.8%, and the estimated total number of diseases in the country was 160 million, an increase of 7,000 from 1991. More than 10,000 people. The number of patients with dyslipidemia in the country has reached 160 million.
  • the inventors have obtained a PCSK9 monoclonal antibody through intensive research and creative labor.
  • the present inventors have surprisingly found that the monoclonal antibody of the present invention binds well to PCSK9 well, and is very effective in blocking the binding of PCSK9 to LDLR, increasing the number of LDLR on the cell surface, and increasing LDL and cholesterol carried.
  • Metabolism which increases triglyceride (TG) metabolism, especially in vivo, has a long-term efficacy and therefore has the potential to be used for the preparation of a medicament for the prevention and treatment of cardiovascular diseases caused by hypercholesterolemia.
  • TG triglyceride
  • One aspect of the invention relates to a monoclonal antibody or antigen-binding fragment thereof, wherein
  • the heavy chain variable region of the monoclonal antibody comprises: a CDR selected from the group consisting of SEQ ID NOs: 5-7;
  • the light chain variable region of the monoclonal antibody comprises an amino acid sequence selected from the group consisting of CDRs of SEQ ID NOs: 8-10.
  • Antibody therapeutics particularly monoclonal antibodies (MABs) have achieved good results in the treatment of a variety of diseases.
  • Traditional experimental methods for obtaining these therapeutic antibodies are to immunize animals with antigens, to obtain antibodies that target antigens in immunized animals, or to improve antibodies that have lower affinity for antigens by affinity maturation.
  • these methods require a lot of time and effort, and most of the time they do not target specific epitopes on the antigen.
  • variable regions of the light and heavy chains determine the binding of the antigen; the variable regions of each chain contain three hypervariable regions, called complementarity determining regions (CDRs) (the CDRs of the heavy chain (H) comprise HCDR1, HCDR2, HCDR3
  • CDRs complementarity determining regions
  • the CDR of the light chain (L) comprises LCDR1, LCDR2, LCDR3; Named by Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition (1991), Vol. 1-3, NIH Publication 91-3242, Bethesda Md).
  • the inventors have creatively constructed the amino acid sequences of the six CDR regions and made specific modifications to increase the binding activity of the antibody variable regions to the antigen.
  • the framework regions are also modified at the same time, but it is necessary to ensure that the modifications of these framework regions are still compatible with human germline sequences. Modifications to the framework will also be analyzed simultaneously to ensure that these changes have no effect on the binding of the CDR regions to the antigen.
  • the three CDRs of the heavy chain variable region of the invention are as follows:
  • HCDR1 GFTFSSYS (SEQ ID NO: 5)
  • HCDR2 ISSSSSYI (SEQ ID NO: 6)
  • HCDR3 EYDFWSAYYDAFDV (SEQ ID NO: 7)
  • the three CDRs of the light chain variable region are as follows:
  • LCDR1 SRNIGGGND (SEQ ID NO: 8)
  • LCDR2 GVI (SEQ ID NO: 9)
  • LCDR3 QSFDGSLSGSV (SEQ ID NO: 10).
  • the monoclonal antibody or antigen-binding fragment thereof wherein
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody is set forth in SEQ ID NO: 2;
  • amino acid sequence of the light chain variable region of the monoclonal antibody is set forth in SEQ ID NO:4.
  • CDRs complementarity determining regions
  • the monoclonal antibody or antigen-binding fragment thereof wherein the monoclonal antibody or antigen-binding fragment thereof is selected from the group consisting of Fab, Fab', F(ab') 2 , Fd, Fv a dAb, a complementarity determining region fragment, a single chain antibody (eg, scFv), a humanized antibody, a chimeric antibody, or a diabody.
  • the monoclonal antibody or antigen-binding fragment thereof wherein the monoclonal antibody is less than about 100 nM, such as less than about 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6nM, 0.5nM, 0.4nM, 0.3nM, 0.2nM, 0.1nM EC 50 or less PCSK9 binding protein; preferably, the EC 50 measured by sandwich ELISA.
  • the monoclonal antibody or antigen-binding fragment thereof wherein the monoclonal antibody is less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or less PCSK9 K D binding protein.
  • the monoclonal antibody or antigen-binding fragment thereof wherein
  • the monoclonal antibody comprises a non-CDR region and the non-CDR region is from a species other than a murine, such as from a human antibody.
  • Another aspect of the invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence capable of encoding an antibody heavy chain variable region, wherein
  • the heavy chain variable region of the antibody comprises: the amino acid sequence selected from the group consisting of the CDRs of SEQ ID NOs: 5-7;
  • the heavy chain variable region of the antibody has the amino acid sequence set forth in SEQ ID NO: 2;
  • the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 1.
  • a further aspect of the invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence capable of encoding a variable region of an antibody light chain, wherein
  • the antibody light chain variable region comprises a CDR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-10;
  • the antibody light chain variable region has the amino acid sequence set forth in SEQ ID NO:4;
  • the nucleic acid molecule has the nucleotide sequence set forth in SEQ ID NO:3.
  • the invention also relates to a vector comprising the isolated nucleic acid molecule of the invention.
  • the invention also relates to a host cell comprising an isolated nucleic acid molecule of the invention, or a vector of the invention.
  • a further aspect of the invention relates to a method of producing a monoclonal antibody or antigen-binding fragment thereof according to any of the invention, which comprises culturing a host cell of the invention under suitable conditions, and recovering from the cell culture The step of the monoclonal antibody or antigen-binding fragment thereof.
  • a further aspect of the invention relates to a conjugate comprising a monoclonal antibody or antigen-binding fragment thereof, and a conjugated portion, wherein the monoclonal antibody is a monoclonal antibody or antigen-binding thereof according to any one of the invention a fragment, the coupling moiety being a detectable label; preferably, the coupling moiety is a radioisotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme.
  • a further aspect of the invention relates to a kit comprising the monoclonal antibody or antigen-binding fragment thereof of any of the invention, or a conjugate of the invention;
  • the kit further comprises a second antibody that specifically recognizes the monoclonal antibody or antigen-binding fragment thereof; optionally, the second antibody further comprises a detectable label, such as a radioisotope, a fluorescent substance , luminescent substances, colored substances or enzymes.
  • a detectable label such as a radioisotope, a fluorescent substance , luminescent substances, colored substances or enzymes.
  • a further aspect of the invention relates to the use of a monoclonal antibody or antigen-binding fragment thereof according to any of the invention or a conjugate of the invention for the preparation of a kit for detecting PCSK9 in a sample The existence or level of it.
  • a further aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof of any one of the invention 5 or a conjugate of the invention; optionally, further comprising a pharmaceutically Acceptable carriers and/or excipients.
  • a further aspect of the present invention relates to the monoclonal antibody or antigen-binding fragment thereof according to any one of the present invention or the conjugate of the present invention for preventing and/or treating hypertension, high cholesterol, hypercholesterolemia and the like
  • the use of drugs for cardiovascular diseases relates to the monoclonal antibody or antigen-binding fragment thereof according to any one of the present invention or the conjugate of the present invention for preventing and/or treating hypertension, high cholesterol, hypercholesterolemia and the like.
  • a further aspect of the invention relates to the use of a monoclonal antibody or antigen-binding fragment thereof according to any of the invention or a conjugate of the invention for the preparation of a medicament as follows:
  • a drug that increases the metabolic level of cholesterol and/or TG carried by LDL is a drug that increases the metabolic level of cholesterol and/or TG carried by LDL.
  • the present inventors also found by experiments (in vivo experiments) that the PCSK9 antibody of the present invention can effectively lower the levels of LDL and/or LDL-C in mouse and monkey plasma, and can be longer than Evolocumab (LDL- in mice) C levels can be reduced by up to 32 days) to lower plasma LDL and / or LDL-C levels.
  • the PCSK9 antibody of the present invention can also reduce serum TG levels in monkeys, and the effect can be maintained for up to 13 days, showing a good application prospect. In the current state of the art, no PCSK9 antibody has been able to achieve this technical effect.
  • a further aspect of the invention relates to a method in vivo or in vitro comprising the step of using an effective amount of the monoclonal antibody of any of the invention or the monoclonal antibody conjugate of the invention, the method being selected As follows:
  • a method of increasing the amount of LDLR on the cell surface or the level of LDLR in plasma is a method of increasing the amount of LDLR on the cell surface or the level of LDLR in plasma
  • a method of increasing the metabolic level of cholesterol and/or TG carried by LDL is a method of increasing the metabolic level of cholesterol and/or TG carried by LDL.
  • a further aspect of the invention relates to a method of treating and/or preventing hypertension, high cholesterol, hypercholesterolemia and cardiovascular diseases thereof, comprising using an effective amount of the invention.
  • the monoclonal antibody or antigen-binding fragment thereof, or the conjugate for use in the prevention and/or treatment of hypertension, high cholesterol, hypercholesterolemia, and the resulting Cardiovascular disease.
  • the monoclonal antibody or antigen-binding fragment thereof, or the conjugate is used in:
  • Specificity binds to PCSK9, blocks PCSK9 binding to LDLR, increases cell surface LDLR levels or plasma LDLR levels, decreases plasma LDL or LDL-C levels, inhibits plasma LDL accumulation, inhibits PCSK9-mediated LDLR degradation, or increases The metabolic level of cholesterol and/or TG carried by LDL.
  • NP_001106130.1 when referring to the amino acid sequence of the PCSK9 protein (Proprotein convertase subtilisin/kexin type 9), it includes not only the full length of the PCSK9 protein (eg, human NP_777596.21, murine NP_705793.1, or monkey source).
  • NP_001106130.1 also includes a fusion protein thereof, such as a fragment fused to a Fc protein fragment (mFc or hFc) of mouse or human IgG.
  • Fc protein fragment mFc or hFc
  • mutations or variations may be naturally occurring or artificially introduced in the amino acid sequence of the PCSK9 protein without affecting its biological function.
  • EC 50 refers to the term as used herein, half-maximal effective concentration (concentration for 50% of maximal effect ), the concentration refers to cause 50% of maximal effect.
  • antibody refers to an immunoglobulin molecule usually composed of two pairs of polypeptide chains (each pair having one "light” (L) chain and one "heavy” (H) chain. .
  • Antibody light chains can be classified as kappa and lambda light chains.
  • Heavy chains can be classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain further comprises a "D" region of about 3 or more amino acids.
  • Each heavy chain is comprised of a heavy chain variable region (V H) and a heavy chain constant region (C H) composition.
  • the heavy chain constant region is comprised of three domains (C H 1, C H 2 and C H 3) components.
  • Each light chain is comprised of a light chain variable region (V L) and a light chain constant region (C L) components.
  • the light chain constant region is comprised of one domain, C L composition.
  • the constant region of the antibody mediates binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system.
  • V H regions may be subdivided into hypervariability regions (termed complementarity determining regions (CDR)), interspersed with regions are more conserved, termed framework regions (FR) of.
  • CDR complementarity determining regions
  • FR framework regions
  • Each V H and V L the following order: FR1, CDR1, FR2, CDR2 , FR3, CDR3, FR4 from the amino terminus to the carboxy terminus arranged three four FR and CDR components.
  • the assignment of amino acids to regions or domains follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J.
  • antibody is not limited by any particular method of producing antibodies. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies.
  • the antibody may be an antibody of a different type, for example, an IgG (eg, IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibody.
  • an antigen-binding fragment of an antibody refers to a polypeptide comprising a fragment of a full length antibody that retains the ability to specifically bind to the same antigen to which the full length antibody binds, and/or compete with the full length antibody.
  • Specific binding to an antigen which is also referred to as an "antigen-binding portion.” See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes.
  • an antigen-binding fragment of an antibody is produced by enzymatic or chemical cleavage of an intact antibody.
  • the antigen-binding fragment includes Fab, Fab', F(ab') 2 , Fd, Fv, dAb and complementarity determining regions (CDRs). Fragments, single chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen binding ability to the polypeptide.
  • Fd fragment means an antibody fragment consisting of V H and C H 1 domains
  • Fv fragment means a single arm of V H and V L domains of an antibody, Antibody fragment
  • dAb fragment means an antibody fragment consisting of a VH domain (Ward et al, Nature 341:544-546 (1989))
  • Fab fragment means by V L , V H , C antibody fragments L and C H 1 domains
  • F (ab ') 2 fragment means antibody fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region.
  • the antigen-binding fragment is a single chain antibody (e.g., the scFv), wherein V L and V H domains are paired to form so that it can be produced by a linker to a single polypeptide chain monovalent molecules (see, e.g., Bird Et al, Science 242: 423-426 (1988) and Huston et al, Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988)).
  • scFv molecules can have the general structure: NH 2 -V L - linker -V H -COOH or NH 2 -V H - linker -V L -COOH.
  • Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or variants thereof.
  • a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448).
  • Other linkers useful in the present invention are by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al. (1996), Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol.
  • the antigen-binding fragments are diabodies, i.e., bivalent antibodies in which V H and V L, domains are expressed on a single polypeptide chain, but using a linker that is too short to not allow the same chain in two Pairing between domains forces the domain to pair with the complementary domain of another strand and create two antigen binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444 -6448 (1993), and Poljak RJ et al., Structure 2: 1121-1123 (1994)).
  • the antigen-binding fragment of the antibody can be obtained from a given antibody using conventional techniques known to those skilled in the art (eg, recombinant DNA techniques or enzymatic or chemical cleavage methods) and in the same manner as used for intact antibodies. Specific screening of antigen-binding fragments of antibodies.
  • antibody As used herein, unless the context clearly dictates otherwise, when referring to the term “antibody”, it includes not only intact antibodies, but also antigen-binding fragments of antibodies.
  • the terms “monoclonal antibody” and “monoclonal antibody” refer to a group of high A fragment of an antibody or antibody in a homologous antibody molecule, that is, a group of identical antibody molecules, except for natural mutations that may occur spontaneously.
  • Monoclonal antibodies are highly specific for a single epitope on the antigen.
  • Polyclonal antibodies are relative to monoclonal antibodies, which typically comprise at least two or more different antibodies, which typically recognize different epitopes on the antigen.
  • Monoclonal antibodies are typically obtained using hybridoma technology first reported by Kohler et al. (Nature, 256:495, 1975), but can also be obtained using recombinant DNA techniques (see, for example, U.S. Patent No. 4,816,567).
  • humanized antibody means that all or part of the CDR regions of a human immunoglobulin (receptor antibody) are replaced by a CDR region of a non-human antibody (donor antibody).
  • An antibody or antibody fragment, wherein the donor antibody can be a non-human (eg, mouse, rat or rabbit) antibody having the desired specificity, affinity or reactivity.
  • some of the amino acid residues of the framework region (FR) of the acceptor antibody can also be replaced by amino acid residues of the corresponding non-human antibody or by amino acid residues of other antibodies to further refine or optimize the performance of the antibody.
  • epitope refers to a site on an antigen that is specifically bound by an immunoglobulin or antibody. "Epitope” is also referred to in the art as an "antigenic determinant.”
  • An epitope or antigenic determinant typically consists of a chemically active surface group of a molecule, such as an amino acid or a carbohydrate or sugar side chain, and typically has specific three dimensional structural characteristics as well as specific charge characteristics.
  • an epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-contiguous amino acids in a unique spatial conformation, which may be "linear" "or” conformational.
  • the term “isolated” or “isolated” refers to a natural Obtained by artificial means. If a certain "separated” substance or component appears in nature, it may be that the natural environment in which it is located has changed, or that the substance has been isolated from the natural environment, or both. For example, a certain living animal has a naturally isolated polynucleotide or polypeptide that is not isolated, and the high purity of the same polynucleotide or polypeptide isolated from this natural state is called separation. of.
  • the term “isolated” or “isolated” does not exclude the inclusion of artificial or synthetic materials, nor does it exclude the presence of other impure substances that do not affect the activity of the material.
  • E. coli expression system refers to an expression system consisting of E. coli (strain) and a vector, wherein E. coli (strain) is derived from a commercially available strain such as, but not limited to, GI698 , ER2566, BL21 (DE3), B834 (DE3), BLR (DE3).
  • vector refers to a nucleic acid vehicle into which a polynucleotide can be inserted.
  • a vector is referred to as an expression vector when the vector enables expression of the protein encoded by the inserted polynucleotide.
  • the vector can be introduced into the host cell by transformation, transduction or transfection, and the genetic material element carried thereby can be expressed in the host cell.
  • Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1 derived artificial chromosomes (PAC).
  • Phage such as lambda phage or M13 phage and animal virus.
  • Animal viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, nipples Multi-tumor vacuolar virus (such as SV40).
  • a vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication.
  • the term "host cell” refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as Escherichia coli or Bacillus subtilis, such as a fungal cell such as a yeast cell or an Aspergillus.
  • a prokaryotic cell such as Escherichia coli or Bacillus subtilis
  • a fungal cell such as a yeast cell or an Aspergillus.
  • S2 Drosophila cells or insect cells such as Sf9
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • an antibody that specifically binds to an antigen means that the antibody is less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, Affinity (K D ) of 10 -8 M, 10 -9 M or 10 -10 M or less binds to the antigen.
  • K D refers to a particular antibody - antigen interaction dissociation equilibrium constant, which is used to describe the binding affinity between antibody and antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.
  • the antibody has a dissociation equilibrium constant (K D ) of less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 M or less.
  • Binding antigen eg, L1 protein
  • SPR surface plasmon resonance
  • amino acids are generally represented by single letter and three letter abbreviations as are known in the art.
  • alanine can be represented by A or Ala.
  • the term "effective amount" refers to an amount sufficient to achieve, or at least partially achieve, a desired effect.
  • a prophylactically effective amount refers to an amount sufficient to prevent, arrest, or delay the onset of the disease; treating an effective amount of the disease means an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Determination of such an effective amount is well within the capabilities of those skilled in the art.
  • the amount effective for therapeutic use will depend on the severity of the condition to be treated, the overall condition of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments for simultaneous administration. and many more.
  • the monoclonal antibody of the present invention (for example, MAB1) can bind specifically to PCSK9, and can effectively block the binding of PCSK9 to LDLR, increase the number of LDLR on the cell surface, increase the metabolism of LDL and the cholesterol carried, and reduce TG level, It has a long-term efficacy in the body and can be used to prevent and reduce cardiovascular diseases caused by hypercholesterolemia.
  • Figure 1 Results of SDS-PAGE detection of PCSK9 antibody MAB1.
  • the left and right lane samples and their loadings were: BSA, 1 ⁇ g; Marker, 10 ⁇ l; Reduced: reduced protein electrophoresis loading buffer sample, 1 ⁇ g; Non-reduced: non-reduced protein electrophoresis loading buffer Sample, 1 ⁇ g.
  • Figure 2 Diagram of the results of the kinetic characteristic parameters of MAB1.
  • Figure 3 ELISA method to detect the binding of MAB1 to human, murine and monkey PCSK9 antibodies, respectively.
  • Figure 4 Competitive binding assay for MAB1 antibody and human PCSK9 antigen to LDLR using a competitive ELISA method.
  • Figure 5 Flow cytometry was used to detect the expression of LDLR on the surface of human liver HepG2 cells after 24 h incubation with MAB1.
  • Figure 6 Flow cytometry was used to detect the expression level of LDLR on the surface of human liver HepG2 cells cultured with MAB1 for 48 hours.
  • Figure 7 SDS-PAGE was used to detect the expression of LDLR on the surface of human liver HepG2 cells after 24 h incubation with MAB1.
  • FIG. 8 Effect of PCSK9 antibodies MAB1 and Evolocumab on serum LDL-C concentration in mice.
  • FIG. 9 Effect of PCSK9 antibody MAB1 on serum LDL-C concentration in monkeys.
  • FIG. 10 Effect of PCSK9 antibodies MAB1 and Evolocumab on serum HDL-C concentration in mice.
  • FIG. 11 Effect of PCSK9 antibody MAB1 on serum HDL-C concentration in monkeys.
  • Figure 12 Effect of PCSK9 antibodies MAB1 and Evolocumab on serum TG concentration in mice.
  • Figure 13 Effect of PCSK9 antibody MAB1 on serum TG concentration in monkeys.
  • the BALB/C mice used were purchased from the Guangdong Medical Laboratory Animal Center.
  • the positive control antibody Evolocumab used was the Amgen antibody Evolocumab (Joyce C. Y. A proprotein convertase subtilisin/kexin type 9 neutralizing antibody reduce serum cholesterol in mice and nonhuman primates. Proc Natl Acad Sci USA, 2009, 106(24): 9820-5).
  • NCBI Reference Sequence: NP_777596.21 The amino acid sequences of human source (NCBI Reference Sequence: NP_777596.21), mouse source (NCBI Reference Sequence: NP_705793.1) and monkey source (NCBI Reference Sequence: NP_001106130.1) were combined with TEV-his6.
  • the nucleic acid sequences corresponding to the obtained fusion proteins were manually optimized, and the corresponding human, mouse and monkey fusion protein genes were synthesized by Kingsray.
  • the synthesized PCSK9-TEV-his6 fusion gene was cloned into the pUC57simple (provided by Kingsray) vector by Kingsray to obtain the pUC57simple-PCSK9-TEV-his6 plasmid.
  • Plasmid Digestion of plasmid pUC57simple-PCSK9-TEV-his6 (Xba I and BamH I), electrophoresis The fusion gene fragment PCSK9-TEV-his was recovered and ligated with the pcDNA3.1 vector to construct pcDNA3.1-PCSK9-TEV-his6 (pcDNA3.1 expression vector was purchased from Invitrogen; DH5a competent was purchased from TIANGEN) And the positive pcDNA3.1-PCSK9-TEV-his6 clone colony was screened, and the E. coli was amplified according to a conventional method and then extracted with a kit to obtain a pcDNA3.1-PCSK9-TEV-his6 recombinant plasmid. (Refer to the kit instructions for the procedure).
  • the human, mouse and monkey-derived recombinant plasmid pcDNA3.1-PCSK9-TEV-his6 constructed above was transfected into 293F cells for 7 days, and then the culture medium was subjected to high-speed centrifugation and microfiltration membrane vacuum filtration, according to the manufacturer.
  • the method of operation was purified by HisTrap column (protein purification liquid chromatography system / AKTA Purifier 10, GE) to obtain purified human, mouse and monkey-derived PCSK9-TEV-his6 fusion protein.
  • the obtained purified fusion protein was digested with TEV protease and purified by a Ni-NTA affinity chromatography column to obtain PCSK9 antigen.
  • the purchased LDLR human cDNA (purchased from Origene) was used as a template for PCR amplification and purified by a common DNA product purification kit to recover the hLDLR-His fragment, and the target fragment was recovered by gel.
  • the recovered target fragment hLDLR-His was digested with XbaI&HindIII-HF, ligated by T4 ligase, and all the ligation products were transformed into DH5a chemically competent cells, Agar plate with Amp was applied, and a single colony with good isolation was selected. Colony PCR identification, clones with positive PCR results were inoculated into LB medium, and the bacteria were sent to Guangzhou Handsome Company for sequencing verification. The sequencing results showed that the positive recombinant insert sequence was completely correct.
  • the recombinant plasmid LDLR-his was transfected into 293F cells (purchased from Invitrogen) for 7 days according to the lipofectamin transfection kit (purchased from Invitrogen), and the culture was centrifuged by high speed, concentrated, and changed to Binding Buffer A. Load to HisTrap For the column, the protein was linearly eluted with Elution Buffer A. The purely pure sample was exchanged with a HiTrap Desalting column to Binding Buffer B and loaded onto a HiTrap Q column. The protein was linearly eluted with Elution Buffer B, and the target sample was recovered and exchanged to PBS. The purified sample was added to a reduced protein electrophoresis loading buffer for SDS-PAGE electrophoresis.
  • the inventors creatively artificially designed a series of antibody sequences based on the PCSK9 protein sequence and its three-dimensional crystal structure. Through extensive screening and detection, a MAB1 specifically binding to PCSK9 was finally obtained.
  • the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody and the coding DNA sequence thereof are set forth in SEQ ID NO: 1-4 below.
  • the heavy chain cDNA sequence of MAB1 (the coding sequence is shown as 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 SEQ ID NO: 3)
  • the constant region was Ig lambda-2 chain C regions; ACCESSION: P0CG05.1) was cloned into pUC57simple (provided by Kingsray) vector to obtain pUCS7simple-MAB1H and pUC57simple-MAB1L plasmids, respectively.
  • the plasmids pUCS7simple-MAB1H and pUC57simple-MAB1L were digested respectively (HindIII & EcoRI), and the heavy chain light chains recovered by electrophoresis were subcloned into pcDNA3.1 vector, and the recombinant plasmid was co-transfected into 293F cells. After 7 days of cell culture, the culture solution was centrifuged by high speed, concentrated, and loaded onto a HiTrap MabSelect SuRe column, and the protein was eluted in one step with Elution Buffer and the target sample was recovered and exchanged to PBS.
  • the purified sample was separately added to a reduced protein electrophoresis loading buffer and a non-reduced protein electrophoresis loading buffer, and then boiled and subjected to SDS-PAGE electrophoresis.
  • MAB1's electricity The swimming chart is shown in Figure 1.
  • the reduced protein sample target protein is at 45 kD and 30 kD
  • the non-reduced protein sample (single antibody) target protein is at 150 kD.
  • the MAB1 produced in this example was used in the following Examples 2-7.
  • the kinetic parameters of MAB1 and human antigen PCSK9-his were determined using a Fortebio molecular interaction instrument.
  • MAB1 (prepared in Example 1) was immobilized on the surface of the AR2G sensor by amino coupling, blocked with ethanolamine, equilibrated in PBST, and combined with antigen PCSK9-his (prepared in Preparation 1), and the antigen was diluted twice with PBST.
  • concentrations were 411, 205.5, 102.8, 51.4, 25.7, 12.8, 0 nM, dissociated in PBST.
  • mouse anti-his monoclonal antibody Jinsrui Bio, A00186
  • BSA mouse antigen serum
  • MAB1 MAB1 for 30 min, added.
  • HRP-labeled goat anti-human IgG secondary antibody Jackson, 109-035-088
  • TMB Neogen, 308177
  • MAB1 efficiently binds to PCSK9 protein and its binding efficiency is dose dependent.
  • MAB1 by antibody binding is the absorbance intensity quantitative analysis, binding efficiency simulated curve EC 50 (Table 6).
  • Table 6 ELISA sandwich assay analysis of the binding efficiency of MAB1 to antigen PCSK9 EC 50
  • the cells were coated with LDLR-his (prepared in Preparation 2) at 4 ° C overnight, and blocked with 1% BSA for 2 h. Then, a mixture of human PCSK9 and MAB1 antigen-antibody (see Table 7 for dilution) was added, and the cells were incubated at 37 ° C for 30 min. The enzyme-labeled secondary antibody was incubated for 1 h and incubated at 37 ° C for 30 min. The absorbance at 450 nm was measured on a microplate reader (Table 7).
  • MAB1 analysis obtained by the light intensity simulation curve of absorbance binding efficiency EC 50 2.339nM. The results clearly show that MAB1 can effectively compete with PCSK9 antigen for binding to LDLR, thereby inhibiting PCSK9-induced decrease in cell surface LDLR (see Example 4 below), which in turn achieves regulation of low-density cholesterol metabolism in vivo (ie, decreased MAB1).
  • the level of low density cholesterol in serum is shown in Example 5) below.
  • LDLR low-density cholesterol receptor
  • Table 8 MAB1 and antigen PCSK9 treatment sample dose table
  • the antigen-antibody was incubated for 24 and 48 hours, respectively, and HepG2 cells were obtained by conventional trypsin digestion and the number of cells in each collection tube was 2 ⁇ 10 5 .
  • PE-labeled rabbit anti-human LDLR was prepared with PBS (containing 1% BSA). The antibody dilution of 1:200) was incubated on ice for 1 hour in the dark, and the cells were washed twice with PBS, 300 ⁇ l of PBS was added, and the fluorescence signal was detected by a PE channel on a flow cytometer.
  • the antibody MAB1 was incubated with the antigen for 24 hours. The results are shown in Figure 5. The results of incubation for 48 hours are shown in Figure 6.
  • Example 5 Effect of MAB1 on the concentration of low density lipoprotein cholesterol (LDL-C) in vivo
  • the blood lipid level before the drug is the normal control.
  • Blood samples were collected before drug administration, 30 min, 5 h, 1 d, 5 d, 7 d, 9 d, 11 d, 13 d, 15 d, 17 d, 19 d, 21 d, and centrifuged at 3000 rpm for 10 min to determine the serum LDL-C content.
  • the equipment used for the determination was Mindray BS-180 automatic biochemical analyzer, and the LDL-C assay kits were purchased from Shenzhen Mindray Biomedical Electronics Co., Ltd.
  • Example 6 Effect of MAB1 on high density lipoprotein cholesterol (HDL-C) concentration in vivo
  • MAB1 120mg/kg administration amount 120mg/kg
  • the blood lipid level before the drug is the normal control.
  • Blood samples were collected before administration, 30 min, 5 h, 1 d, 5 d, 7 d, 9 d, 11 d, 13 d, 15 d, 17 d, 19 d, 21 d, and centrifuged at 3000 rpm for 10 min to determine the serum HDL-C content.
  • the equipment used for the measurement was Mindray BS-180 automatic biochemical analyzer, and the HDL-C assay kits were purchased from Shenzhen Mindray Biomedical Electronics Co., Ltd.
  • Blood samples were collected before administration, 30 min, 5 h, 1 d, 5 d, 7 d, 9 d, 11 d, 13 d, 15 d, 17 d, 19 d, 21 d, and centrifuged at 3000 rpm for 10 min to determine the serum TG content.
  • the equipment used for the determination was Mindray BS-180 automatic biochemical analyzer, and the TG assay kits were purchased from Shenzhen Mindray Biomedical Electronics Co., Ltd.

Abstract

本发明提供了一种PCSK9的单克隆抗体，其能够特异性结合PCSK9并阻断PCSK9与LDLR的结合。本发明的单克隆抗体可用于防治和减少高胆固醇血症所引起的心血管疾病。

Description

PCSK9抗体、其药物组合物及其用途 技术领域
本发明属于免疫学和分子生物学领域，涉及一种PCSK9抗体、其药物组合物及其用途。具体地，本发明涉及PCSK9的单克隆抗体。
背景技术
前蛋白转化酶枯草杆菌蛋白酶Kexin-9型(Proprotein convertase subtilisin/kexin type 9，PCSK9)是一种具有前蛋白转化酶活性的枯草溶菌素蛋白酶。PCSK9在肝脏、小肠和肾脏中表达量较多，在皮肤、神经系统中有少量表达。肝脏中的PCSK9可以分泌到血循环中，并通过结合与内吞作用降低细胞表面低密度脂蛋白受体(LDL receptor，LDLR)的数目。因为LDLR可以从血浆中高效消除LDL，LDLR受体数量减少可促使LDL积聚，所以PCSK9所介导的LDLR降解，可以提高血浆低密度脂蛋白胆固醇(LDL-C)的水平。PCSK9参与富含甘油三脂的载脂蛋白B的脂代谢，PCSK9的分泌能提高肠道甘油三脂(TG)的水平，从而参与诱发高胆固醇血症(Rashid S.，et al.，Proprotein convertase subtilisin kexin type 9 promotes intestinal overproduction of triglyceride-rich apolipoprotein B lipoproteins through both low-density lipoprotein receptor-dependent and-independent mechanisms.Circulation.2014 Jul 29；130(5)：431-41.)。
高胆固醇血症病人中有29％的病人在使用他汀类药物。不能耐受他汀类药物和他汀类药物不能达到所需血脂胆固醇水平的患者占高胆固醇血症病人的8.2％。全球七大药物市场(不包括中国)共有高胆固醇血症病人3亿9千万。2004年10月12日，卫生部公布的《中国居民营养与健康状况调查》报告显示，我国成人高血压患病率为18.8％，估计全国患病总数为1.6亿人，比1991年增加了7000多万人。全国血脂异常患者也已达到1.6亿人。
目前，尚需要开发新的能够特异作用于人类PCSK9的免疫治疗抗 体，其可结合至PCSK9并阻断PCSK9与LDLR的结合。阻断PCSK9与LDLR的结合可以提高细胞表面LDLR的数量，提高LDL和所携带胆固醇的代谢，防治和减少高胆固醇血症所引起的心血管疾病。尤其需要开发高效能的PCSK9抗体，其还能够同时提高甘油三脂(TG)代谢或降低TG水平，甚至能够在较长的时间内维持功效。
发明内容
本发明人经过深入的研究和创造性的劳动，得到了一种PCSK9单克隆抗体。本发明人惊奇地发现，本发明的单克隆抗体能够很好地特异性与PCSK9结合，并且能够十分有效地阻断PCSK9与LDLR的结合，提高细胞表面LDLR的数量，提高LDL和所携带胆固醇的代谢，提高甘油三脂(TG)代谢，特别是在体内的药效时间长，因此具有用于制备防治高胆固醇血症所引起的心血管疾病的药物的潜力。由此提供了下述发明：
本发明的一个方面涉及一种单克隆抗体或其抗原结合片段，其中，
所述单克隆抗体的重链可变区包含：氨基酸序列选自SEQ ID NO：5-7的CDR；
和/或
所述单克隆抗体的轻链可变区包含：氨基酸序列选自SEQ ID NO：8-10的CDR。
抗体治疗药物，特别是单克隆抗体(MAB)已在多种疾病的治疗中取得了良好的疗效。获取这些治疗性抗体的传统实验方法是采用抗原免疫动物，在免疫动物体内获取靶向抗原的抗体，或通过亲和力成熟的方法来改进那些与抗原的亲和力较低的抗体。然而，这些方法需要大量时间和精力，大多数时候也并不能针对抗原上的特定表位。
轻链和重链的可变区决定抗原的结合；每条链的可变区均含有三个高变区，称互补决定区(CDR)(重链(H)的CDR包含HCDR1、HCDR2、HCDR3，轻链(L)的CDR包含LCDR1、LCDR2、LCDR3；其由 Kabat等人命名，见Sequences of Proteins of Immunological Interest，Fifth Edition(1991)，第1-3卷，NIH Publication 91-3242，Bethesda Md)。
本发明人创造性地构建了6个CDR区的氨基酸序列，并做特定修改以提高抗体可变区对抗原的结合活性。为配合在CDR区的改动，框架区也同时被修改，但是需要确保这些框架区的修改仍与人源的种系序列相兼容。框架的修改也将同时进行分析，以确保这些改变对CDR区与抗原的结合没有影响。
本发明重链可变区的3个CDR如下：
HCDR1：GFTFSSYS  (SEQ ID NO：5)
HCDR2：ISSSSSYI  (SEQ ID NO：6)
HCDR3：EYDFWSAYYDAFDV  (SEQ ID NO：7)
轻链可变区的3个CDR如下：
LCDR1：SRNIGGGND  (SEQ ID NO：8)
LCDR2：GVI  (SEQ ID NO：9)
LCDR3：QSFDGSLSGSV  (SEQ ID NO：10)。
在本发明的一些实施方案中，所述的单克隆抗体或其抗原结合片段，其中，
所述单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO：2所示；
和/或
所述单克隆抗体的轻链可变区的氨基酸序列如SEQ ID NO：4所示。
通过本领域技术人员所熟知的技术手段，例如通过美国国立生物技术信息中心网站(NCBI)，分析该单克隆抗体重链可变区和轻链可变区的互补决定区(CDR)，其亦分别如上面的SEQ ID NO：5-7和SEQ ID NO：8-10所示。在本发明中，将该单克隆抗体命名为MAB1。
在本发明的一些实施方案中，所述的单克隆抗体或其抗原结合片段，其中，所述单克隆抗体或其抗原结合片段选自Fab、Fab′、F(ab′)2、 Fd、Fv、dAb、互补决定区片段、单链抗体(例如，scFv)、人源化抗体、嵌合抗体或双抗体。
在本发明的一些实施方案中，所述的单克隆抗体或其抗原结合片段，其中，所述的单克隆抗体以小于大约100nM，例如小于大约10nM、1nM、0.9nM、0.8nM、0.7nM、0.6nM、0.5nM、0.4nM、0.3nM、0.2nM、0.1nM或更小的EC50结合PCSK9蛋白；优选地，所述EC50通过ELISA夹心法测得。
在本发明的一些实施方案中，所述的单克隆抗体或其抗原结合片段，其中，所述的单克隆抗体以小于大约10-5M，例如小于大约10-6M、10-7M、10-8M、10-9M或10-10M或更小的KD结合PCSK9蛋白。
在本发明的一些实施方案中，所述的单克隆抗体或其抗原结合片段，其中，
所述的单克隆抗体包括非-CDR区，且所述非-CDR区来自不是鼠类的物种，例如来自人抗体。
本发明的另一方面涉及分离的核酸分子，其包含能够编码抗体重链可变区的核酸序列，其中，
所述抗体的重链可变区包含：氨基酸序列选自SEQ ID NO：5-7的CDR；
优选地，所述抗体的重链可变区具有SEQ ID NO：2所示的氨基酸序列；
更优选地，所述核酸分子具有SEQ ID NO：1所示的核苷酸序列。
本发明的再一方面涉及分离的核酸分子，其包含能够编码抗体轻链可变区的核酸序列，其中，
所述抗体轻链可变区包含氨基酸序列选自SEQ ID NO：8-10的CDR；
优选地，所述抗体轻链可变区具有SEQ ID NO：4所示的氨基酸序列；
更优选地，所述核酸分子具有SEQ ID NO：3所示的核苷酸序列。
本发明还涉及一种载体，其包含本发明的分离的核酸分子。
本发明还涉及一种宿主细胞，其包含本发明的分离的核酸分子，或者本发明的载体。
本发明的再一方面涉及制备本发明中任一项所述的单克隆抗体或其抗原结合片段的方法，其包括在合适的条件下培养本发明的宿主细胞，以及从细胞培养物中回收所述单克隆抗体或其抗原结合片段的步骤。
本发明的再一方面涉及偶联物，其包括单克隆抗体或其抗原结合片段以及偶联部分，其中，所述单克隆抗体为本发明中任一项所述的单克隆抗体或其抗原结合片段，所述偶联部分为可检测的标记；优选地，所述偶联部分为放射性同位素、荧光物质、发光物质、有色物质或酶。
本发明的再一方面涉及试剂盒，其包括本发明中任一项所述的单克隆抗体或其抗原结合片段，或者包括本发明的偶联物；
优选地，所述试剂盒还包括第二抗体，其特异性识别所述单克隆抗体或其抗原结合片段；任选地，所述第二抗体还包括可检测的标记，例如放射性同位素、荧光物质、发光物质、有色物质或酶。
本发明的再一方面涉及本发明中任一项所述的单克隆抗体或其抗原结合片段或者本发明的偶联物在制备试剂盒中的用途，所述试剂盒用于检测PCSK9在样品中的存在或其水平。
本发明的再一方面涉及一种药物组合物，其包含本发明5中任一项所述的单克隆抗体或其抗原结合片段或者本发明的偶联物；可选地，其还包括药学上可接受的载体和/或赋形剂。
本发明的再一方面涉及本发明中任一项所述的单克隆抗体或其抗原结合片段或者本发明的偶联物在制备预防和/或治疗高血压、高胆固醇、高胆固醇血症及其所引起的心血管疾病的药物中的用途。
本发明的再一方面涉及本发明中任一项所述的单克隆抗体或其抗原结合片段或者本发明的偶联物在制备如下药物中的用途：
特异性与PCSK9结合的药物，
阻断PCSK9与LDLR结合的药物，
提高细胞表面LDLR数量或者血浆中LDLR水平的药物，
降低血浆中LDL或LDL-C水平的药物，
抑制血浆中LDL积聚的药物，
抑制PCSK9所介导的LDLR降解的药物，或者
提高LDL所携带胆固醇和/或TG的代谢水平的药物。
本发明人还通过实验(体内实验)发现，本发明的PCSK9抗体能够有效地降低小鼠和猴血浆中LDL和/或LDL-C水平，并且相比Evolocumab能更长时间(小鼠体内LDL-C水平可降低达32天)降低血浆中LDL和/或LDL-C水平。特别是出乎意料地发现，本发明的PCSK9抗体还能降低猴体内血清的TG水平，并且效果能够维持长达13天，显示了良好的应用前景。目前现有技术中，尚未有任何PCSK9抗体能够实现这一技术效果。
本发明的再一方面涉及一种在体内或体外的方法，包括使用有效量的本发明中任一项所述的单克隆抗体或者本发明的单克隆抗体偶联物的步骤，所述方法选自如下：
特异性与PCSK9结合的方法，
阻断PCSK9与LDLR结合方法，
提高细胞表面LDLR数量或者血浆中LDLR水平的方法，
降低血浆中LDL或LDL-C水平的方法，
抑制血浆中LDL积聚的方法，
抑制PCSK9所介导的LDLR降解的方法，或者
提高LDL所携带胆固醇和/或TG的代谢水平的方法。
本发明的再一方面涉及一种治疗和/或预防高血压、高胆固醇、高胆固醇血症及其所引起的心血管疾病的方法，包括使用有效量的本发明中 任一项所述的单克隆抗体或者本发明的单克隆抗体偶联物的步骤。
在本发明的一些实施方案中，所述的单克隆抗体或其抗原结合片段或者所述的偶联物，其用于预防和/或治疗高血压、高胆固醇、高胆固醇血症及其所引起的心血管疾病。
在本发明的一些实施方案中，所述的单克隆抗体或其抗原结合片段或者所述的偶联物，其用于：
特异性与PCSK9结合、阻断PCSK9与LDLR结合、提高细胞表面LDLR数量或者血浆中LDLR水平、降低血浆中LDL或LDL-C水平、抑制血浆中LDL积聚、抑制PCSK9所介导的LDLR降解或者提高LDL所携带胆固醇和/或TG的代谢水平。
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，当提及PCSK9蛋白(Proprotein convertase subtilisin/kexin type 9)的氨基酸序列时，其不仅包括PCSK9蛋白的全长(例如人源NP_777596.21、鼠源NP_705793.1或猴源NP_001106130.1)，还包括其融合蛋白，例如与小鼠或人IgG的Fc蛋白片段(mFc或hFc)进行融合的片段。然而，本领域技术人员理解，在PCSK9蛋白的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于置换，缺失和/或添加)，而不影响其生物学功能。
如本文中所使用的，术语EC50是指半最大效应浓度(concentration for 50％of maximal effect)，是指能引起50％最大效应的浓度。
如本文中所使用的，术语“抗体”是指，是指通常由两对多肽链(每对具有一条“轻”(L)链和一条“重”(H)链)组成的免疫球蛋白分子。抗体轻链可分类为κ和λ轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链 内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(vH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗体结合部位。氨基酸至各区域或结构域的分配遵循Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health，Bethesda，Md.(1987 and 1991))，或Chothia & Lesk(1987)J.Mol.Biol.196：901-917；Chothia等人(1989)Nature 342：878-883的定义。术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，特别地，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
如本文中所使用的，术语抗体的“抗原结合片段”是指包含全长抗体的片段的多肽，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合，其也被称为“抗原结合部分”。通常参见，Fundamental Immunology，Ch.7(Paul，W.，ed.，第2版，Raven Press，N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。在一些情况下，抗原结合片段包括Fab、Fab′、F(ab′)2、Fd、Fv、dAb和互补决定区(CDR)片段、单链抗体(例如，scFv)、嵌合抗体、双抗体(diabody)和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。
如本文中所使用的，术语“Fd片段”意指由VH和CH1结构域组成的 抗体片段；术语“Fv片段”意指由抗体的单臂的VL和VH结构域组成的抗体片段；术语“dAb片段”意指由VH结构域组成的抗体片段(Ward等人，Nature 341：544-546(1989))；术语“Fab片段”意指由VL、VH、CL和CH1结构域组成的抗体片段；术语“F(ab′)2片段”意指包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段。
在一些情况下，抗体的抗原结合片段是单链抗体(例如，scFv)，其中VL和VH结构域通过使其能够产生为单个多肽链的连接体配对形成单价分子(参见，例如，Bird等人，Science 242：423-426(1988)和Huston等人，Proc.Natl.Acad.Sci.USA 85：5879-5883(1988))。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS)4的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90：6444-6448)。可用于本发明的其他接头由Alfthan等人(1995)，Protein Eng.8：725-731，Choi等人(2001)，Eur.J.Immunol.31：94-106，Hu等人(1996)，Cancer Res.56：3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293：41-56和Roovers等人(2001)，Cancer Immunol.描述。
在一些情况下，抗体的抗原结合片段是双抗体，即，双价抗体，其中VH和VL结构域在单个多肽链上表达，但使用太短的连接体以致不允许在相同链的两个结构域之间配对，从而迫使结构域与另一条链的互补结构域配对并且产生两个抗原结合部位(参见，例如，Holliger P.等人，Proc.Natl.Acad.Sci.USA 90：6444-6448(1993)，和Poljak R.J.等人，Structure 2：1121-1123(1994))。
可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的抗体获得抗体的抗原结合片段，并且以与用于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合片段。
在本文中，除非上下文明确指出，否则当提及术语“抗体”时，其不仅包括完整抗体，而且包括抗体的抗原结合片段。
如本文中所使用的，术语“单抗”和“单克隆抗体”是指，来自一群高 度同源的抗体分子中的一个抗体或抗体的一个片断，也即除可能自发出现的自然突变外，一群完全相同的抗体分子。单抗对抗原上的单一表位具有高特异性。多克隆抗体是相对于单克隆抗体而言的，其通常包含至少2种或更多种的不同抗体，这些不同的抗体通常识别抗原上的不同表位。单克隆抗体通常可采用Kohler等首次报道的杂交瘤技术获得(Nature，256：495，1975)，但也可采用重组DNA技术获得(如参见U.S.P4,816,567)。
如本文中所使用的，术语“人源化抗体”是指，人源免疫球蛋白(受体抗体)的全部或部分CDR区被一非人源抗体(供体抗体)的CDR区替换后得到的抗体或抗体片段，其中的供体抗体可以是具有预期特异性、亲和性或反应性的非人源(例如，小鼠、大鼠或兔)抗体。此外，受体抗体的构架区(FR)的一些氨基酸残基也可被相应的非人源抗体的氨基酸残基替换，或被其他抗体的氨基酸残基替换，以进一步完善或优化抗体的性能。关于人源化抗体的更多详细内容，可参见例如，Jones et al.，Nature，321：522 525(1986)；Reichmann et al.，Nature，332：323 329(1988)；Presta，Curr.Op.Struct.Biol.，2：593 596(1992)；和Clark，Immunol.Today 21：397 402(2000)。
如本文中所使用的，术语“表位”是指，抗原上被免疫球蛋白或抗体特异性结合的部位。“表位”在本领域内也称为“抗原决定簇”。表位或抗原决定簇通常由分子的化学活性表面基团例如氨基酸或碳水化合物或糖侧链组成并且通常具有特定的三维结构特征以及特定的电荷特征。例如，表位通常以独特的空间构象包括至少3，4，5，6，7，8，9，10，11，12，13，14或15个连续或非连续的氨基酸，其可以是“线性的”或“构象的”。参见，例如，Epitope Mapping Protocols in Methods in Molecular Biology，第66卷，G.E.Morris，Ed.(1996)。在线性表位中，蛋白质与相互作用分子(例如抗体)之间的所有相互作用的点沿着蛋白质的一级氨基酸序列线性存在。在构象表位中，相互作用的点跨越彼此分开的蛋白质氨基酸残基而存在。
如本文中所使用的，术语“分离的”或“被分离的”指的是，从天然状 态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”或“被分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。
如本文中所使用的，术语“大肠杆菌表达系统”是指由大肠杆菌(菌株)与载体组成的表达系统，其中大肠杆菌(菌株)来源于市场上可得到的菌株，例如但不限于：GI698，ER2566，BL21(DE3)，B834(DE3)，BLR(DE3)。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
如本文中使用的，术语“特异性结合”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。在某些实施方式中，特异性结合某抗原的抗体(或对某抗原具有特异性的抗体)是指，抗体以小于大约10-5M，例如小于大约10-6M、10-7M、10-8M、10-9M或10-10M或更小的亲和力(KD)结合该抗原。
如本文中所使用的，术语“KD”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。通常，抗体以小于大约10-5M，例如小于大约10-6M、10-7M、10-8M、10-9M或10-10M或更小的解离平衡常数(KD)结合抗原(例如，L1蛋白)，例如，如使用表面等离子体共振术(SPR)在BIACORE仪中测定的。
如本文中所使用的，术语“单克隆抗体”和“单抗”具有相同的含义且可互换使用；术语“多克隆抗体”和“多抗”具有相同的含义且可互换使用；术语“多肽”和“蛋白质”具有相同的含义且可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病有效量是指，足以预防，阻止，或延迟疾病的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
发明的有益效果
本发明的单克隆抗体(例如MAB1)能够很好地特异性与PCSK9结合，并且能够十分有效地阻断PCSK9与LDLR的结合，提高细胞表面LDLR的数量，提高LDL和所携带胆固醇的代谢，降低TG水平， 在体内的药效时间长，可用于防治和减少高胆固醇血症所引起的心血管疾病。
附图说明
图1：PCSK9抗体MAB1的SDS-PAGE检测结果。从左至右的泳道样品及其上样量依次为：BSA，1μg；Marker，10μl；Reduced：还原型蛋白电泳上样缓冲液样品，1μg；Non-reduced：非还原型蛋白电泳上样缓冲液样品，1μg。
图2：MAB1的动力学特征参数检测结果图。
图3：ELISA方法检测MAB1分别与人、鼠、猴PCSK9抗体结合的曲线图。
图4：采用竞争ELISA方法检测MAB1抗体及人源PCSK9抗原与LDLR的竞争结合。
图5：流式细胞术检测MAB1孵育人肝HepG2细胞24h后细胞表面LDLR的表达水平。
图6：流式细胞术检测MAB1孵育人肝HepG2细胞48h后细胞表面LDLR的表达水平。
图7：SDS-PAGE检测MAB1孵育人肝HepG2细胞24h后细胞表面LDLR的表达水平。
图8：PCSK9抗体MAB1和Evolocumab对小鼠体内血清LDL-C浓度的影响。
图9：PCSK9抗体MAB1对猴体内血清LDL-C浓度的影响。
图10：PCSK9抗体MAB1和Evolocumab对小鼠体内血清HDL-C浓度的影响。
图11：PCSK9抗体MAB1对猴体内血清HDL-C浓度的影响。
图12：PCSK9抗体MAB1和Evolocumab对小鼠体内血清TG浓度的影响。
图13：PCSK9抗体MAB1对猴体内血清TG浓度的影响。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或按照产品说明书进行。所用试剂或仪器未注明生产厂商者，为可以通过市场购买获得的常规产品。
在本发明的下述实施例中，使用的BALB/C小鼠购自广东省医学实验动物中心。
所用的阳性对照抗体Evolocumab是Amgen抗体Evolocumab(Joyce C.Y.A proproteinconvertasesubtilisin/kexin type 9 neutralizing antibody reduces serum cholesterol in mice and nonhuman primates.ProcNatlAcadSci USA，2009，106(24)：9820-5)。
制备例1：人源、鼠源和猴源的PCSK9-TEV-his6融合蛋白的设计 和制备
1.基因PCSK9-TEV-his6的合成
分别对人源(NCBI Reference Sequence：NP_777596.21)、鼠源(NCBI Reference Sequence：NP_705793.1)和猴源(NCBI Reference Sequence：NP_001106130.1)PCSK9氨基酸序列与TEV-his6进行融合设计。分别将获得的融合蛋白所对应的核酸序列进行人工优化，并委托金斯瑞公司合成优化后的相对应的人源、鼠源和猴源融合蛋白基因。
2.pUC57simple-PCSK9-TEV-his6质粒的获得
由金斯瑞公司将合成的PCSK9-TEV-his6融合基因克隆到pUC57simple(金斯瑞公司提供)载体中，获得pUC57simple-PCSK9-TEV-his6质粒。
pcDNA3-PCSK9-TEV-his6重组质粒的构建：将质粒pUC57simple-PCSK9-TEV-his6进行酶切后(Xba I和BamH I)，电泳 回收得到的融合基因片段PCSK9-TEV-his并与pcDNA3.1载体进行连接反应重组构建获得pcDNA3.1-PCSK9-TEV-his6(pcDNA3.1表达载体购自Invitrogen公司；DH5a感受态购自TIANGEN公司)，并筛选得到阳性的pcDNA3.1-PCSK9-TEV-his6克隆菌落，按照常规方法扩增大肠杆菌然后采用试剂盒提取得到pcDNA3.1-PCSK9-TEV-his6重组质粒。(操作步骤参照试剂盒说明书)。
3.融合蛋白PCSK9-TEV-his6的表达、纯化及抗原PCSK9的获得
将上面构建的人源、鼠源和猴源的重组质粒pcDNA3.1-PCSK9-TEV-his6分别转染293F细胞7天后，将培养液进行高速离心、微孔滤膜抽真空过滤，根据厂家提供的操作方法采用HisTrap柱(蛋白纯化液相色谱系统/AKTA Purifier 10，GE)进行纯化，分别得到纯化的人源、鼠源和猴源PCSK9-TEV-his6融合蛋白。将得到的纯化的融合蛋白分别用TEV蛋白酶进行酶切，并过Ni-NTA亲和层析柱纯化，获得PCSK9抗原。
制备例2：LDLR蛋白的表达和纯化
1.构建hLDLR-His质粒
以购买的LDLR human cDNA(购自Origene公司)为模板进行PCR扩增并用普通DNA产物纯化试剂盒纯化回收hLDLR-His片段，并凝胶回收目的片段。将回收后的目的片段hLDLR-His与XbaI&HindIII-HF酶切，通过T4连接酶连接，并将全部连接产物转化DH5a化学感受态细胞，涂布带有Amp的Agar平板，挑选分离良好的单菌落进行菌落PCR鉴定，将PCR鉴定结果为阳性的克隆子接种到LB培养基培养，并取菌液送广州英俊公司测序验证，测序结果比对显示，阳性重组子插入序列完全正确。
2.LDLR蛋白的表达和纯化
按照lipofectamin转染试剂盒(购自Invitrogen公司)方法将重组质粒LDLR-his转染293F细胞(购自Invitrogen公司)7天后，将培养液通过高速离心、上清浓缩并换液至Binding Buffer A后上样至HisTrap 柱，用Elution Buffer A线性洗脱蛋白，初纯样品用HiTrap Desalting柱换液至Binding Buffer B并上样至HiTrap Q柱，用Elution Buffer B线性洗脱蛋白、回收目标样品并换液至PBS。取纯化后样品加入还原型蛋白电泳上样缓冲液，进行SDS-PAGE电泳检测。
实施例1：MAB1重链和轻链序列的设计，表达和纯化
1.抗体的设计
为制备单克隆抗体MAB1，本发明人根据PCSK9蛋白序列及其蛋白三维晶体结构等，创造性地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。该单克隆抗体重链可变区和轻链可变区的氨基酸序列及其编码DNA序列如下面的SEQ ID NO：1-4。
编码单克隆抗体MAB1重链可变区的核酸序列(369 bp)：
Figure PCTCN2016073492-appb-000001
单克隆抗体MAB1重链可变区的氨基酸序列(123 aa)：
Figure PCTCN2016073492-appb-000002
编码单克隆抗体MAB1轻链可变区的核酸序列(333 bp)：
Figure PCTCN2016073492-appb-000003
单克隆抗体MAB1轻链可变区的氨基酸序列(111 aa)：
Figure PCTCN2016073492-appb-000004
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pUCS7simple-MAB1H和pUC57simple-MAB1L质粒。
分别将质粒pUCS7simple-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。
将纯化后的样品分别加入还原型蛋白电泳上样缓冲液和非还原型蛋白电泳上样缓冲液，煮沸后进行SDS-PAGE电泳检测。MAB1的电 泳图如图1所示，还原型蛋白样品目标蛋白在45kD和30kD处，非还原型蛋白样品(单个抗体)目标蛋白在150kD处。
本实施例制得的MAB1用于下面的实施例2-7。
实施例2：MAB1与抗原PCSK9-his的动力学参数测定
使用Fortebio分子相互作用仪测定MAB1与人源抗原PCSK9-his的动力学参数。
MAB1(实施例1制得)采用氨基偶联的方式固定于AR2G传感器表面，经乙醇胺封闭，于PBST中平衡后，与抗原PCSK9-his(制备例1制备)结合，抗原用PBST两倍稀释，浓度为411、205.5、102.8、51.4、25.7、12.8、0nM，于PBST中解离。
MAB1的动力学参数见表2，检测结果如图2所示。
表2：MAB1的动力学参数
抗体名称 KD(M) Kon(l/Ms) kon误差 Kdis(l/s) kdis误差
MAB1 7.19E-10 2.00E+05 2.08E+03 1.44E-04 5.11E-06
KD为亲和力常数；kon为抗原抗体结合速率；kdis为抗原抗体解离速率；KD＝kdis/kon。
结果表明，抗体MAB1与抗原有较好的亲和力。
实施例3：MAB1与抗原PCSK9的结合活性分析
1.采用ELISA夹心法分别测定单克隆抗体与鼠源、猴源和人源抗原PCSK9(制备例1制备)的结合活性。
将包被鼠抗his单克隆抗体(金斯瑞生物，A00186)的酶标板用BSA封闭2h，分别加入鼠源、猴源和人源抗原PCSK9预保温30min，分别加入MAB1进行孵育30min，加入HRP标记羊抗人IgG二抗(Jackson，109-035-088)，用TMB(Neogen，308177)进行显色反应5min，并在酶标仪中检测450nm波长吸光度。
检测结果如图3所示。
由图3可见，MAB1能有效地结合PCSK9蛋白，并且其结合效率呈剂量依赖关系。
MAB1抗体不同稀释浓度梯度结合鼠源、猴源和人源抗原PCSK9的吸光强度见表3-表5。
表3：MAB1与鼠源PCSK9的结合(ELISA夹心法)
Figure PCTCN2016073492-appb-000005
表4：MAB1与猴源PCSK9的结合(ELISA夹心法)
Figure PCTCN2016073492-appb-000006
Figure PCTCN2016073492-appb-000007
表5：MAB1与人源PCSK9的结合(ELISA夹心法)
Figure PCTCN2016073492-appb-000008
Figure PCTCN2016073492-appb-000009
通过对结合的MAB1抗体进行吸光强度定量分析，曲线模拟得到结合效率EC50(表6)。
表6：ELISA夹心法分析MAB1与抗原PCSK9的结合效率EC50
Figure PCTCN2016073492-appb-000010
结果表明，MAB1能有效地结合人、鼠和猴PCSK9。
2.采用竞争ELISA方法测定MAB1及PCSK9抗原(人源)与LDLR的竞争结合。
用LDLR-his(制备例2制得)包被酶标板4℃过夜，1％BSA封闭2h后加入人源PCSK9和MAB1抗原抗体混合液(稀释浓度见表7)，37℃孵育30min后加入酶标二抗孵育1h，37℃孵育30min。在酶标仪上检测450nm的吸光值(表7)。
检测MAB1与LDLR(制备例2制备)竞争结合的结果如图4所示。
由图4可见，MAB1有效地阻断抗原PSCK9与LDLR的结合，并且其结合效率呈剂量依赖关系。
各剂量的吸光强度见表7。结果表明，抗体MAB1能有效地结合PCSK9蛋白并抑制PCSK9诱导的细胞表面LDLR下降(即MAB1可以有效阻断PCSK9与LDLR的结合及后者的内吞噬现象)，并且这种抑制作用与抗体剂量呈正相关性(即剂量依赖关系)。
表7：MAB1抗体与PSCK9抗原竞争LDLR结合
Figure PCTCN2016073492-appb-000011
Figure PCTCN2016073492-appb-000012
通过吸光强度曲线模拟分析得到MAB1的结合效率EC50为2.339nM。结果很明显地表明，MAB1能有效地与PCSK9抗原竞争结合LDLR，从而抑制PCSK9所诱导的细胞表面LDLR下降(见后面的实施例4)，继而达到低密度胆固醇体内代谢的调节作用(即MAB1降低血清中低密度胆固醇的水平，见后面的实施例5)。
实施例4：MAB1的细胞生物学活性分析
为检测MAB1对人肝细胞株HepG2细胞中低密度胆固醇受体(LDLR)水平的调节作用，采用流式方法和蛋白免疫印迹方法检测MAB1对HepG2细胞表面LDLR水平的调节作用。
1.流式细胞法检测MAB1对HepG2细胞LDLR水平的调节作用
取生长良好的HepG2细胞样本，移去旧培养液，按如下设计加抗原抗体分别孵育24h和48h。
表8：MAB1及抗原PCSK9处理样本剂量表
Figure PCTCN2016073492-appb-000013
抗原抗体分别孵育24，48小时后，采用常规胰酶消化方法获取HepG2细胞并使每个收集管细胞数为2×105，用PBS(含1％BSA)配制PE标记的兔抗人LDLR(1∶200)的抗体稀释液，冰上避光孵育1小时后，用PBS洗涤细胞2次后加入300μl PBS，在流式细胞仪上用PE通道检测荧光信号。
抗体MAB1与抗原孵育24h检测结果如图5所示。孵育48h检测结果如图6所示。
由图5、图6可见，MAB1呈剂量依赖性阻断PCSK9引发的细胞表面LDLR表达的降低，能有效地拮抗抗原PCSK9对LDLR的负性调节作用。
2.蛋白免疫印迹法检测MAB1对人干细胞株HepG2细胞表面LDLR水平的调节作用
取生长良好的HepG2细胞样本，移去旧培养基，按表8设计加抗体药物孵育24h。药物处理后，收集并裂解细胞，对收集的裂解上清进行SDS-PAGE电泳检测，如图7所示，目标蛋白在～140kD处。检测结果显示：MAB1有效地增加了LDLR的表达水平，并呈现剂量依赖的关系；并且有效地拮抗抗原PCSK9-His对LDLR的负性调节作用。
实施例5：MAB1对体内低密度脂蛋白胆固醇(LDL-C)浓度的影响
1.MAB1对小鼠血清LDL-C浓度的影响
为检测MAB1对小鼠血清LDL-C浓度的影响，取实验小鼠分为4组进行皮下注射：Control(生理盐水组，给药方式为10mL/kg，n＝8)， MAB1 60mg/kg(给药量60mg/kg，给药方式为10mg/kg，n＝6)，MAB1 90mg/kg(给药量90mg/kg，给药方式为20mg/kg，n＝6)，Evolocumab 60mg/kg(给药量60mg/kg，给药方式为10mg/kg，n＝4)。分别在药前、给药3d、7d、10d、18d、24d、32d后眼眶采血150μl，4500rpm离心10min分离血清，分别在给药3d、7d、10d、18d、24d、32d测定血清中低密度脂蛋白胆固醇(LDL-C)含量。测定所用设备为迈瑞BS-180全自动生化分析仪，LDL-C测定试剂盒均自深圳迈瑞生物医疗电子股份有限公司。
结果如图8所示。结果表明，抗PCSK9抗体MAB1皮下注射给药3天后能够明显降低BALB/c小鼠血清LDL-C的含量，与Evolocumab药效相当；MAB1的药效时间(达32天)比Evolocumab药效作用时间(18天)长。
2.MAB1对猴血清LDL-C浓度的影响
为检测MAB1对猴血清LDL-C浓度的影响，将4只雄性健康食蟹猴分为2组：MAB13mg/kg组(n＝2)和MAB118mg/kg组(n＝2)，皮下注射给药，以药前血脂水平为正常对照。分别在药前、给药30min、5h、1d、5d、7d、9d、11d、13d、15d、17d、19d、21d采血，3000rpm离心10min分离血清，测定血清中LDL-C含量。测定所用设备为迈瑞BS-180全自动生化分析仪，LDL-C测定试剂盒均购自深圳迈瑞生物医疗电子股份有限公司。
结果显示，抗PCSK9抗体MAB1的剂量分别为3mg/kg和18mg/kg均能够明显降低食蟹猴血清LDL-C的含量，18mg/kg给药剂量的药效维持时间长达17天(图9)。
实施例6：MAB1对体内高密度脂蛋白胆固醇(HDL-C)浓度的影响
1.MAB1对小鼠血清HDL-C浓度的影响
为检测MAB1对小鼠血清HDL-C浓度的影响，取实验小鼠分为5组进行皮下注射：Control(生理盐水组，给药方式为10mL/kg，n＝8)，MAB1 60mg/kg(给药量60mg/kg，给药方式为10mg/kg，n＝6)，MAB1  90mg/kg(给药量90mg/kg，给药方式为20mg/kg，n＝6)，MAB1 120mg/kg(给药量120mg/kg，给药方式为20mg/kg，n＝3)，Evolocumab 60mg/kg(给药量60mg/kg，给药方式为10mg/kg，n＝4)。分别在药前、给药3d、7d、10d、18d、24d、32d后眼眶采血150μl，4500rpm离心10min分离血清，分别在给药3d、7d、10d、18d、24d、32d测定血清中高密度脂蛋白胆固醇(HDL-C)含量。测定所用设备为迈瑞BS-180全自动生化分析仪，HDL-C测定试剂盒均自深圳迈瑞生物医疗电子股份有限公司。
结果如图10所示。结果表明，抗PCSK9抗体MAB1和Evolocumab药效相当，皮下注射给药3天后能够降低BALB/c小鼠血清HDL-C的含量，10天后HDL-C的含量恢复到对照组水平。
2.MAB1对猴血清HDL-C浓度的影响
为检测MAB1对猴血清HDL-C浓度的影响，将4只雄性健康食蟹猴分为2组：MAB13mg/kg组(n＝2)和MAB118mg/kg组(n＝2)，皮下注射给药，以药前血脂水平为正常对照。分别在药前、给药30min、5h、1d、5d、7d、9d、11d、13d、15d、17d、19d、21d采血，3000rpm离心10min分离血清，测定血清中HDL-C含量。测定所用设备为迈瑞BS-180全自动生化分析仪，HDL-C测定试剂盒均购自深圳迈瑞生物医疗电子股份有限公司。
结果显示，抗PCSK9抗体MAB1的剂量分别为3mg/kg和18mg/kg均对食蟹猴血清HDL-C的含量无显著影响(图11)。
实施例7：MAB1对体内甘油三酯(TG)浓度的影响
1.检测MAB1对小鼠血清TG浓度的影响
为检测MAB1对小鼠血清TG浓度的影响，取实验小鼠分为4组进行皮下注射：Control(生理盐水组，给药方式为10mL/kg，n＝8)，MAB1 60mg/kg(给药量60mg/kg，给药方式为10mg/kg，n＝6)，MAB1 90mg/kg(给药量90mg/kg，给药方式为20mg/kg，n＝6)，Evolocumab 60mg/kg(给药量60mg/kg，给药方式为10mg/kg，n＝4)。分别在药前、 给药3d、7d、10d、18d、24d、32d后眼眶采血150μl，4500rpm离心10min分离血清，分别在给药3d、7d、10d、18d、24d、32d测定血清中TG含量。测定所用设备为迈瑞BS-180全自动生化分析仪，LDL-C测定试剂盒均自深圳迈瑞生物医疗电子股份有限公司。
结果如图12所示。结果表明，抗PCSK9抗体MAB1皮下注射给药3天后在高剂量(90mg/kg)能够提高BALB/c小鼠血清TG的含量，给药32天后剂量为60mg/kg能够提高BALB/c小鼠血清TG的含量，其余时间点和Control组没有显著性差异。
2.检测MAB1对猴血清TG浓度的影响
将4只雄性健康食蟹猴分为2组：MAB1 3mg/kg组(n＝2)和MAB1 18mg/kg组，皮下注射给药，以药前血脂水平为正常对照。分别在药前、给药30min、5h、1d、5d、7d、9d、11d、13d、15d、17d、19d、21d采血，3000rpm离心10min分离血清，测定血清中TG含量。测定所用设备为迈瑞BS-180全自动生化分析仪，TG测定试剂盒均购自深圳迈瑞生物医疗电子股份有限公司。
结果显示，抗PCSK9抗体MAB1的剂量为18mg/kg时能够明显降低食蟹猴血清TG的含量，药效维持时间可达13天(图13)。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (20)

  1. 单克隆抗体或其抗原结合片段，其中，
    所述单克隆抗体的重链可变区包含：氨基酸序列选自SEQ ID NO：5-7的CDR；
    和/或
    所述单克隆抗体的轻链可变区包含：氨基酸序列选自SEQ ID NO：8-10的CDR。
  2. 根据权利要求1所述的单克隆抗体或其抗原结合片段，其中，
    所述单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO：2所示；
    和/或
    所述单克隆抗体的轻链可变区的氨基酸序列如SEQ ID NO：4所示。
  3. 根据权利要求1至2中任一权利要求所述的单克隆抗体或其抗原结合片段，其中，所述单克隆抗体或其抗原结合片段选自Fab、Fab′、F(ab′)2、Fd、Fv、dAb、互补决定区片段、单链抗体(例如，scFv)、人源化抗体、嵌合抗体或双抗体。
  4. 根据权利要求1至2中任一权利要求所述的单克隆抗体或其抗原结合片段，其中，所述的单克隆抗体以小于大约10-5M，例如小于大约10-6M、10-7M、10-8M、10-9M或10-10M或更小的KD结合PCSK9蛋白。
  5. 根据权利要求1至2中任一权利要求所述的单克隆抗体或其抗原结合片段，其中，
    所述的单克隆抗体包括非-CDR区，且所述非-CDR区来自不是鼠 类的物种，例如来自人抗体。
  6. 分离的核酸分子，其包含能够编码抗体重链可变区的核酸序列，其中，
    所述抗体的重链可变区包含：氨基酸序列选自SEQ ID NO：5-7的CDR；
    优选地，所述抗体的重链可变区具有SEQ ID NO：2所示的氨基酸序列；
    更优选地，所述核酸分子具有SEQ ID NO：1所示的核苷酸序列。
  7. 分离的核酸分子，其包含能够编码抗体轻链可变区的核酸序列，其中，
    所述抗体轻链可变区包含氨基酸序列选自SEQ ID NO：8-10的CDR；
    优选地，所述抗体轻链可变区具有SEQ ID NO：4所示的氨基酸序列；
    更优选地，所述核酸分子具有SEQ ID NO：3所示的核苷酸序列。
  8. 一种载体，其包含权利要求6和/或7所述的分离的核酸分子。
  9. 一种宿主细胞，其包含权利要求6和/或7所述的分离的核酸分子，或者权利要求8所述的载体。
  10. 制备权利要求1至5中任一权利要求所述的单克隆抗体或其抗原结合片段的方法，其包括在合适的条件下培养权利要求9的宿主细胞，以及从细胞培养物中回收所述单克隆抗体或其抗原结合片段的步骤。
  11. 偶联物，其包括单克隆抗体或其抗原结合片段以及偶联部分，其中，所述单克隆抗体为权利要求1至5中任一权利要求所述的单克隆 抗体或其抗原结合片段，所述偶联部分为可检测的标记；优选地，所述偶联部分为放射性同位素、荧光物质、发光物质、有色物质或酶。
  12. 试剂盒，其包括权利要求1至5中任一权利要求所述的单克隆抗体或其抗原结合片段，或者包括权利要求11所述的偶联物；
    优选地，所述试剂盒还包括第二抗体，其特异性识别所述单克隆抗体或其抗原结合片段；任选地，所述第二抗体还包括可检测的标记，例如放射性同位素、荧光物质、发光物质、有色物质或酶。
  13. 权利要求1至5中任一权利要求所述的单克隆抗体或其抗原结合片段或者权利要求11所述的偶联物在制备试剂盒中的用途，所述试剂盒用于检测PCSK9在样品中的存在或其水平。
  14. 一种药物组合物，其包含权利要求1至5中任一权利要求所述的单克隆抗体或其抗原结合片段或者权利要求11所述的偶联物；可选地，其还包括药学上可接受的载体和/或赋形剂。
  15. 权利要求1至5中任一权利要求所述的单克隆抗体或其抗原结合片段或者权利要求11所述的偶联物在制备预防和/或治疗高血压、高胆固醇、高胆固醇血症及其所引起的心血管疾病的药物中的用途。
  16. 权利要求1至5中任一权利要求所述的单克隆抗体或其抗原结合片段或者权利要求11所述的偶联物在制备如下药物中的用途：
    特异性与PCSK9结合的药物，
    阻断PCSK9与LDLR结合的药物，
    提高细胞表面LDLR数量或者血浆中LDLR水平的药物，
    降低血浆中LDL或LDL-C水平的药物，
    抑制血浆中LDL积聚的药物，
    抑制PCSK9所介导的LDLR降解的药物，或者
    提高LDL所携带胆固醇和/或TG的代谢水平的药物。
  17. 一种在体内或体外的方法，包括使用有效量的权利要求1至5中任一权利要求所述的单克隆抗体或者权利要求11所述的单克隆抗体偶联物的步骤，所述方法选自如下：
    特异性与PCSK9结合的方法，
    阻断PCSK9与LDLR结合方法，
    提高细胞表面LDLR数量或者血浆中LDLR水平的方法，
    降低血浆中LDL或LDL-C水平的方法，
    抑制血浆中LDL积聚的方法，
    抑制PCSK9所介导的LDLR降解的方法，或者
    提高LDL所携带胆固醇和/或TG的代谢水平的方法。
  18. 一种治疗和/或预防高血压、高胆固醇、高胆固醇血症及其所引起的心血管疾病的方法，包括使用有效量的权利要求1至5中任一权利要求所述的单克隆抗体或者权利要求11所述的单克隆抗体偶联物的步骤。
  19. 权利要求1至5中任一权利要求所述的单克隆抗体或其抗原结合片段或者权利要求11所述的偶联物，其用于预防和/或治疗高血压、高胆固醇、高胆固醇血症及其所引起的心血管疾病。
  20. 权利要求1至5中任一权利要求所述的单克隆抗体或其抗原结合片段或者权利要求11所述的偶联物，其用于：
    特异性与PCSK9结合、阻断PCSK9与LDLR结合、提高细胞表面LDLR数量或者血浆中LDLR水平、降低血浆中LDL或LDL-C水平、抑制血浆中LDL积聚、抑制PCSK9所介导的LDLR降解或者提高LDL所携带胆固醇和/或TG的代谢水平。
PCT/CN2016/073492 2015-02-11 2016-02-04 Pcsk9抗体、其药物组合物及其用途 WO2016127912A1 (zh)

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