WO2017114230A1 - Pcsk9抗体、其抗原结合片段及其医药用途 - Google Patents

Pcsk9抗体、其抗原结合片段及其医药用途 Download PDF

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WO2017114230A1
WO2017114230A1 PCT/CN2016/111053 CN2016111053W WO2017114230A1 WO 2017114230 A1 WO2017114230 A1 WO 2017114230A1 CN 2016111053 W CN2016111053 W CN 2016111053W WO 2017114230 A1 WO2017114230 A1 WO 2017114230A1
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seq
variable region
chain variable
heavy chain
light chain
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PCT/CN2016/111053
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English (en)
French (fr)
Inventor
屈向东
叶鑫
徐韶瑜
袁蓓
崔东冰
胡齐悦
张蕾
许志宾
陶维康
张连山
孙飘扬
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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Priority to MX2018007925A priority Critical patent/MX2018007925A/es
Priority to EP16881019.0A priority patent/EP3398968B1/en
Priority to US16/066,567 priority patent/US10793643B2/en
Priority to RU2018125003A priority patent/RU2739208C2/ru
Priority to KR1020187020978A priority patent/KR20180093068A/ko
Priority to BR112018013256-0A priority patent/BR112018013256A2/zh
Application filed by 江苏恒瑞医药股份有限公司, 上海恒瑞医药有限公司 filed Critical 江苏恒瑞医药股份有限公司
Priority to CA3009904A priority patent/CA3009904A1/en
Priority to AU2016382932A priority patent/AU2016382932B2/en
Priority to CN201680013073.1A priority patent/CN107406511B/zh
Priority to JP2018533863A priority patent/JP7032662B2/ja
Publication of WO2017114230A1 publication Critical patent/WO2017114230A1/zh
Priority to HK18104378.4A priority patent/HK1244828A1/zh

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    • 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
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • 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
    • 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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • 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

Definitions

  • the present invention relates to a PCSK9 antibody, an antigen-binding fragment of a PCSK9 antibody, a chimeric antibody comprising the CDR region of the PCSK9 antibody, a humanized antibody, and a pharmaceutical composition comprising the PCSK9 antibody and antigen-binding fragment thereof, and The use of blood lipid drugs.
  • Hypercholesterolemia is a disorder of lipid metabolism characterized by elevated serum cholesterol levels, which is mainly caused by elevated serum cholesterol levels, leading to cholesterol accumulation in blood vessels and atherosclerosis.
  • a large number of clinical and experimental studies have confirmed that lipid metabolism abnormalities are closely related to the occurrence and development of coronary heart disease. Therefore, lowering the concentration of cholesterol in the blood has become a major means of treating and preventing atherosclerosis.
  • the main lipid-lowering drugs are mainly statins.
  • Liptor the world's most widely used cholesterol-lowering drug, is also the best-selling drug in the history of medicine. By blocking the enzymes that produce cholesterol in the liver, it reduces cholesterol production and increases the liver's intake of more cholesterol from the blood. , thereby reducing the concentration of cholesterol in the blood.
  • Lipitor also has its shortcomings. First, from the data, Lipitor can reduce LDL by 30%-40%, but there are still many patients still unable to reach effective blood lipid lowering (low-density lipoprotein concentration ⁇ 50mg/dL), the second patient's response rate to Lipitor is also different. For these reasons, patients need a more effective drug to lower blood lipids.
  • FM Familial hypercholesterolemia
  • LDL-c low density lipoprotein-cholesterol
  • Low-density lipoprotein receptor (LDLR) gene mutation caused defects or deficiency, LDL-c could not be smoothly transported to the liver to clear, resulting in elevated blood LDL-c levels.
  • LDLR gene apolipoprotein B100 gene
  • PCSK9 proprotein convertase subtilisin/kexin type 9
  • PCSK9 The proprotein convertase subtilisin 9 (PCSK9) is a proprotein convertase belonging to the proteinase K subfamily of the secreted subtilisin family.
  • the encoded protein is synthesized as a soluble zymogen and processed in the endoplasmic reticulum by autocatalytic intramolecular processing.
  • the results showed that PCSK9 promoted the degradation of LDL receptors and increased plasma LDL cholesterol content, while LDL receptors mediate LDL endocytosis in the liver, which is the main pathway for clearing LDL from the circulatory system.
  • ADH hypercholesterolemia
  • PCSK9 mutations are diverse in form and can be divided into two categories based on the different effects of mutations on the regulation of LDK-C levels by PCSK9: loss of function and function acquisition. Among them, loss-of-function mutations are associated with low blood cholesterol levels and prevent coronary atherosclerotic heart disease. The rate of PCSK9 mutations in low cholesterol in African populations is higher than in other races.
  • PCSK9 function-acquired mutants increase plasma cholesterol levels by increasing PCSK9 function and decreasing LDLR expression, leading to severe hypercholesterolemia and premature coronary atherosclerotic heart disease, and currently found PCSK9 function-acquired mutations Including: D374Y, S127R, F216L, N157K, R306S and so on. Among them, compared with PCSK9 wild type, the LDLR on the surface of D374Y mutant cells was reduced by 36%, and the S127R mutation was reduced by 10%.
  • PCSK9 as a potential, new target has become a hot spot in the study of hypercholesterolemia, which is of great significance for understanding the mechanism of cholesterol metabolism and seeking new treatment methods.
  • a number of multinational pharmaceutical companies have developed monoclonal antibodies against PCSK9, which neutralize PCSK9 in the blood, thereby increasing the concentration of LDL receptors on the liver surface, thereby reducing the concentration of LDL in the blood.
  • the present invention provides PCSK9 antibodies with higher affinity, higher selectivity, and higher biological activity.
  • the invention provides a PCSK9 antibody or antigen-binding fragment thereof comprising one or more CDRs selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14, or SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14 has an HCDR of the sequence having at least 95% sequence identity; and as of SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: An LCDR as shown by the sequence of 17 or having at least 95% sequence identity to SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17.
  • the PCSK9 antibody or antigen-binding fragment thereof of the invention comprises HCDR1 as set forth in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively.
  • the PCSK9 antibody or antigen-binding fragment thereof of the present invention comprises the LCDR1 as shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, respectively.
  • the above amino acid sequence having at least 95% sequence identity can be obtained by mutating the CDR regions of the present invention by means of affinity maturation.
  • the PCSK9 antibody or antigen-binding fragment thereof of the present invention wherein the antibody or antigen-binding fragment thereof is a murine antibody or a fragment thereof.
  • the PCSK9 antibody or antigen-binding fragment thereof of the present invention wherein the light chain variable region of the PCSK9 antibody further comprises a light of a murine ⁇ chain or a murine ⁇ chain variant a light chain FR region of a chain FR region, or a murine lambda chain or a murine lambda chain variant; wherein the heavy chain variable region of the PCSK9 antibody further comprises a heavy chain FR region of murine IgG1 or a variant thereof, or IgG2 Or a heavy chain FR region thereof, or a heavy chain FR region of IgG3 or a variant thereof.
  • the PCSK9 antibody or antigen-binding fragment thereof of the present invention wherein the murine antibody comprises the heavy chain variable region sequence of SEQ ID NO: 10 and SEQ ID NO: Light chain variable region sequence.
  • the PCSK9 antibody or antigen-binding fragment thereof of the present invention wherein the light chain of the PCSK9 antibody further comprises a light chain constant region of a murine kappa chain or a variant thereof, or a mouse a light chain constant region of a source lambda chain or variant thereof; wherein the PCSK9 antibody heavy chain further comprises a heavy chain constant region of murine IgG1 or a variant thereof, or a heavy chain constant region of IgG2 or a variant thereof, or IgG3 or The heavy chain constant region of its variant.
  • the PCSK9 antibody or antigen-binding fragment thereof of the present invention wherein the antibody or antigen-binding fragment thereof is a chimeric antibody or a fragment thereof.
  • the PCSK9 antibody or antigen-binding fragment thereof of the present invention wherein the antibody or antigen-binding fragment thereof is a humanized antibody or a fragment thereof.
  • the PCSK9 antibody or antigen-binding fragment thereof of the present invention wherein the heavy chain FR region sequence on the heavy chain variable region of the humanized antibody is derived from human germline a combination sequence of a chain, IGHV1-2*02 and hjh2, and a mutant sequence thereof; the FR1 region of the FR1, FR2, FR3 region and hjh2 of the human germline heavy chain IGHV1-2*02 and its mutated sequence, or at least 95% sequence identity amino acid sequence.
  • the PCSK9 antibody or antigen-binding fragment thereof of the present invention wherein the humanized antibody comprises the heavy chain variable region of SEQ ID NO: 18; or SEQ ID a heavy chain variable region of the NO:18 variant; wherein the SEQ ID NO:18 variant is a sequence having a 0-10 amino acid change at the position of the heavy chain variable region set forth in SEQ ID NO:18 .
  • the amino acid change may be improved by improving the properties of the antibody such as affinity, half-life, etc. by using the prior art, such as affinity ripening. Modify the amino acids of the CDR regions or modify the amino acids of the FR region with a back mutation.
  • the PCSK9 antibody or antigen-binding fragment thereof of the present invention wherein the humanized antibody heavy chain FR region sequence has a back mutation of 0-10 amino acids, preferably one Or a plurality of amino acid back mutations selected from the group consisting of T30N, R87T, R72A, T74K, M48I, V68A, M70L, R38K and R67K.
  • the PCSK9 antibody or antigen-binding fragment thereof of the present invention wherein the humanized antibody comprises SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21.
  • the PCSK9 antibody or antigen-binding fragment thereof of the present invention wherein the light chain FR region sequence on the light chain variable region of the humanized antibody is derived from human light a combination sequence of the strand template IGKV1-39*01 and hjk2.1 and a mutant sequence thereof; the FR1 region of the FR1 region, the FR3 region of hzk2.1 and the mutation sequence thereof of the human germline light chain IGKV1-39*01, Or an amino acid sequence with at least 95% sequence identity thereto.
  • the PCSK9 antibody or antigen-binding fragment thereof of the present invention wherein the humanized antibody further comprises the variant of SEQ ID NO: 24 or the variant of SEQ ID NO: 24.
  • the light chain variable region is shown; the SEQ ID NO: 24 variant is an amino acid change having a 0-10 position at the light chain variable region position set forth in SEQ ID NO:24.
  • the amino acid change may be an improvement in the performance of an antibody such as affinity, half-life, etc., using prior art techniques, such as modifying the amino acid of the CDR region with affinity maturation, or modifying the amino acid of the FR region with a back mutation.
  • the PCSK9 antibody or antigen-binding fragment thereof of the present invention wherein the variant of SEQ ID NO: 24 is the light chain variable region of SEQ ID NO:
  • the FR region has a back mutation of 0-10 amino acids; preferably, the back mutation is selected from the amino acid back mutation of T5S, S66D, Q3V and A49S; preferably the amino acid change of A43S.
  • the PCSK9 antibody or antigen-binding fragment thereof of the present invention wherein the humanized antibody comprises SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: The light chain variable region shown by the sequence of 27.
  • the PCSK9 antibody or antigen-binding fragment thereof of the present invention wherein the humanized antibody comprises a heavy chain variable region sequence and/or a light chain variable region sequence
  • the heavy chain variable region sequence is selected from the group consisting of the sequences set forth in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23, or the heavy chain variable region
  • the sequence is selected from sequences having at least 95% sequence identity to SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23;
  • the sequence is selected from the sequences set forth in SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27, or the light chain variable region sequence is selected from the group consisting of SEQ ID NO: 25, SEQ ID NO: And SEQ ID NO: 27 have at least 95% sequence identity sequence.
  • the PCSK9 antibody or antigen-binding fragment thereof of the present invention wherein the PCSK9 antibody comprises a heavy chain variable region and a light chain variable region selected from the group consisting of:
  • the PCSK9 antibody or antigen-binding fragment thereof of the present invention wherein the heavy chain of the PCSK9 antibody further comprises a heavy chain of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof a constant region, or an amino acid sequence having at least 95% sequence identity thereto; preferably comprising a human heavy chain constant region of an IgGl, IgG2 or IgG4 variant comprising human IgGl, IgG2 or IgG4 or using an amino acid mutation to increase the half-life of the antibody in serum, More preferably, an IgG1, IgG2 or IgG4 heavy chain constant region comprising a YTE mutation is introduced;
  • the light chain of the PCSK9 antibody further comprises a constant region of a human kappa, a lambda chain or a variant thereof, or an amino acid sequence having at least 95% sequence identity thereto.
  • the PCSK9 antibody or antigen-binding fragment thereof of the present invention wherein the humanized antibody comprises a heavy chain and a light chain selected from the group consisting of:
  • the invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a PCSK9 antibody or antigen-binding fragment thereof as described above, together with one or more pharmaceutically acceptable carriers, diluents or excipients.
  • the invention further provides a DNA molecule encoding a PCSK9 antibody or antigen-binding fragment thereof as described above.
  • the present invention further provides an expression vector for a DNA molecule as described above.
  • the invention further provides a host cell transformed with an expression vector as described above, the host cell being selected from the group consisting of a prokaryotic cell and a eukaryotic cell, preferably a eukaryotic cell, more preferably a mammalian cell.
  • the invention further provides a PCSK9 antibody or antigen-binding fragment thereof as described above, or a pharmaceutical composition as described above, for use in the manufacture of a medicament for the treatment of a PCSK9 mediated disease or condition, wherein the disease or
  • the condition is preferably a cholesterol-related disease (which includes "serum cholesterol-related diseases"); more preferably hypercholesterolemia, heart disease, metabolic syndrome, diabetes, coronary heart disease, stroke, cardiovascular disease, Alzheimer's disease And general dyslipidemia; most preferred are hypercholesterolemia, dyslipidemia, atherosclerosis, CVD or coronary heart disease.
  • Exemplary diseases that can be diagnosed using the antibodies of the present invention include cholesterol-related diseases (including "serum cholesterol-related diseases") including any one or more of the following: hypercholesterolemia, heart disease, metabolic syndrome, diabetes , coronary heart disease, stroke, cardiovascular disease, Alzheimer's disease, and general dyslipidemia (shown as, for example, increased total serum cholesterol, elevated LDL, increased triglycerides, and extremely low elevation) Density lipoprotein (VLDL) and/or low HDL).
  • VLDL Density lipoprotein
  • the invention provides methods of treating or preventing hypercholesterolemia and/or at least one of the following symptoms in an individual: dyslipidemia, atherosclerosis, cardiovascular disease (CVD) or coronary heart disease
  • the method comprises administering to the individual an effective amount of an anti-PCSK9 antibody.
  • the invention also provides the use of an effective amount of an anti-PCSK9 antibody antagonizing extracellular or circulating PCSK9 for the preparation of a medicament for the treatment or prevention of hypercholesterolemia and/or at least one of the following symptoms in an individual: abnormal lipemia Symptoms, atherosclerosis, CVD or coronary heart disease.
  • Figure 1 Schematic diagram of primer design in the construction of the antibody vector of the present invention.
  • FIG. 1 Schematic diagram of the construction of the antibody vector of the present invention.
  • FIG. 3 Changes in LDL uptake of HepG2 cells in different h001-4-YTE anti-PCSK9 antibody concentrations. The data showed that PCSK9 antibody can promote the uptake of LDL by HepG2 cells.
  • FIG. 4 Changes in LDL uptake of HepG2 cells in different h001-4-WT anti-PCSK9 antibody concentrations. The data showed that PCSK9 antibody can promote the uptake of LDL by HepG2 cells.
  • FIG. 5 LDL-c concentration in mouse serum injected with h001-4-WT anti-PCSK9 antibody as a function of time (*: p ⁇ 0.05, vs IgG, **: p ⁇ 0.01, vs IgG).
  • the data showed that the PCSK9 antibody was able to reduce the concentration of LDL-c in the serum of mice overexpressing human PCSK9.
  • Figure 6 Changes in LDL-c concentration in the relative IgG group of mouse sera injected with h001-4-WT anti-PCSK9 antibody. The data showed that the PCSK9 antibody was able to reduce the LDL-c concentration in the serum of mice overexpressing human PCSK9 relative to the IgG group.
  • Figure 7 Pharmacodynamic and pharmacological detection of the antibody of the present invention in cynomolgus monkeys.
  • the figure shows that h001-4-WT and h001-4-YTE can significantly reduce the LDL content in cynomolgus monkeys, and the duration of h001-4-YTE reduction is better than h001-4-WT.
  • the "antibody” as used in the present invention refers to an immunoglobulin, which is a tetrapeptide chain structure in which two identical heavy chains and two identical light chains are linked by interchain disulfide bonds.
  • the immunoglobulin heavy chain constant region has different amino acid composition and arrangement order, so its antigenicity is also different. Accordingly, immunoglobulins can be classified into five classes, or isoforms of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and the corresponding heavy chains are ⁇ chain, ⁇ chain, and ⁇ chain, respectively. , ⁇ chain, and ⁇ chain.
  • IgG can be classified into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified as either a kappa chain or a lambda chain by the constant region.
  • Each class Ig of the five classes of Ig may have a kappa chain or a lambda chain.
  • the antibody light chain variable region of the present invention may further comprise a light chain constant region comprising a human or murine kappa, lambda chain or a variant thereof.
  • the antibody heavy chain variable region of the present invention may further comprise a heavy chain constant region comprising human or murine IgG1, IgG2, IgG3, IgG4 or a variant thereof.
  • variable region The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly, being the variable region (Fv region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (Fc region).
  • the variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining region (CDR).
  • CDR complementarity determining region
  • Each of the light chain variable region (LCVR) and the heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, and the order from the amino terminus to the carboxy terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • the CDR amino acid residues of the LCVR region and the HCVR region of the antibody or antigen-binding fragment of the present invention conform to the known Kabat numbering rules (LCDR1-3, HCDE2-3) in number and position, or conform to the numbering rules of kabat and chothia. (HCDR1).
  • the antibody of the present invention includes a murine antibody, a chimeric antibody, a humanized antibody, preferably a humanized antibody.
  • murine antibody is in the present invention for human PCSK9 prepared according to the knowledge and skill in the art. Monoclonal antibodies. The test subject is injected with the PCSK9 antigen at the time of preparation, and then the hybridoma expressing the antibody having the desired sequence or functional properties is isolated.
  • the murine PCSK9 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a murine ⁇ , ⁇ chain or a variant thereof, or further comprising a murine IgG1, IgG2 The heavy chain constant region of IgG3 or a variant thereof.
  • chimeric antibody is an antibody obtained by fusing a variable region of a murine antibody with a constant region of a human antibody, and can alleviate an immune response induced by a murine antibody.
  • a hybridoma that secretes a murine-specific monoclonal antibody is selected, and then the variable region gene is cloned from the mouse hybridoma cell, and the constant region gene of the human antibody is cloned as needed to change the mouse.
  • the region gene and the human constant region gene are ligated into a chimeric gene and inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system.
  • the antibody light chain of the PCSK9 chimeric antibody further comprises a light chain Fc region of a human kappa, lambda chain or variant thereof.
  • the antibody heavy chain of the PCSK9 chimeric antibody further comprises a heavy chain Fc region of human IgG1, IgG2, IgG3, IgG4 or variants thereof, preferably comprising a human IgG1, IgG2 or IgG4 heavy chain constant region, or using an amino acid mutation
  • An IgGl, IgG2 or IgG4 variant that extends the half-life of the antibody in serum eg, a YTE mutation.
  • humanized antibody also known as CDR-grafted antibody, refers to the transplantation of mouse CDR sequences into human antibody variable region frameworks, ie different types of human germline An antibody produced in an antibody framework sequence. It is possible to overcome the strong antibody variable antibody response induced by chimeric antibodies by carrying a large amount of mouse protein components.
  • framework sequences can be obtained from public DNA databases including germline antibody gene sequences or published references.
  • the germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk/vbase ), as well as in Kabat, EA, etc.
  • humanized antibodies of the invention also include humanized antibodies that are further affinity matured by phage display.
  • the CDR sequence of the PCSK9 humanized antibody mouse is selected from the group consisting of SEQ ID NO: 12, 13, 14, 15, 16 or 17;
  • the human antibody variable region framework is designed Selected, wherein the light chain FR region sequence on the variable region of the antibody light chain is derived from the combined sequence of human germline light chain IGKV1-39*01 and hjk2.1; wherein the antibody is on the heavy chain variable region
  • the heavy chain FR region sequence is derived from the combined sequence of the human germline heavy chains IGHV1-2*02 and hjh2.
  • the human antibody variable region can be subjected to minimal reverse mutation to maintain activity.
  • the "antigen-binding fragment” as used in the present invention refers to a Fab fragment having antigen-binding activity, a Fab' fragment, a F(ab') 2 fragment, and an Fv fragment ScFv fragment which binds to human PCSK9; NO: 12 to CDR regions of one or more of the antibodies of the invention in SEQ ID NO: 17.
  • the Fv fragment contains the antibody heavy chain variable region and the light chain variable region, but has no constant region and has the smallest antibody fragment of the entire antigen binding site.
  • Fv antibodies also comprise a polypeptide linker between the VH and VL domains and are capable of forming the desired structure for antigen binding.
  • the two antibody variable regions can also be joined by a different linker into a single polypeptide chain, referred to as a single chain antibody or a single chain Fv (sFv).
  • binding to PCSK9 in the present invention means that it is capable of interacting with human PCSK9.
  • antigen binding site refers to a three-dimensional spatial site that is discrete on an antigen and is recognized by an antibody or antigen-binding fragment of the present invention.
  • Fc region is used herein to define a C-terminal region of an immunoglobulin heavy chain that comprises at least a portion of a constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carbonyl terminus of the heavy chain.
  • the C-terminal lysine (Lys447) of the Fc region may or may not be present.
  • the numbering of amino acid residues in the Fc region or constant region is based on the EU numbering system, which is also referred to as the EU index, as in Kabat et al., Sequences of Proteins of Immunological Interest. ), 5th Ed.
  • the Fc region is required for the effector function of the antibody. Effector functions include initiation of complement-dependent cytotoxicity (CDC), initiation of phagocytosis, and antibody-dependent cell-mediated cytotoxicity (ADCC) and transport of antibodies through the cellular barrier by transcytosis. Furthermore, the Fc region is critical for maintaining the serum half-life of IgG class antibodies (Ward and Ghetie, Ther. Immunol. 2: 77-94 (1995)). Studies have found that the serum half-life of IgG antibodies is mediated by the binding of Fc to the neonatal Fc receptor (FcRn).
  • FcRn neonatal Fc receptor
  • FcRn is a heterodimer composed of a transmembrane alpha chain and a soluble beta chain (beta2-microglobulin).
  • U.S. Patent No. 6,165,745 discloses a method of producing a biological half-life reducing antibody by introducing a mutation into a DNA fragment encoding the antibody. This mutation includes amino acid substitutions at positions 253, 310, 311, 433 or 434 of the Fc-strand domain.
  • 6,277,375 B1 discloses a composition comprising a mutant IgG molecule having an increased half-life relative to wild-type IgG, wherein the mutant IgG molecule comprises the following amino acid substitutions: substitution of leucine at position 252 for leucine, at 254 The threonine is substituted for serine, or the phenylalanine at position 256 is substituted for phenylalanine (M252Y, S254T and T256E). Mutant IgGs having amino acid substitutions at positions 433, 435 or 436 are also disclosed.
  • 6,528,624 discloses a variant of an antibody comprising an IgG Fc region at one or more amino acid positions in the human IgG Fc region (positions 270, 322, 326, 327, 329, 331, 333 and 334). ) has an amino acid substitution.
  • WO 02/060919 A2 discloses modified IgG comprising an IgG constant region comprising one or more amino acid modifications relative to a wild-type IgG constant region, wherein the modified IgG is associated with a wild type IgG constant region
  • the half-life is increased compared to IgG, and one or more of the amino acid modifications are located at one or more of the following positions: 251, 253, 255, 285-290, 308-314, 385-389, and 428-435.
  • "YTE” or "YET mutation” as used herein refers to a mutated combination of the Fc region of IgGl for promoting binding of the Fc region to human FcRn, prolonging the half-life of the antibody in human serum.
  • the YTE mutant comprises a combination of three "YTE mutants": M252Y, S254T and T256E, the residue numbering being according to the EU numbering system, which is also referred to as the EU index, as described in Kabat et al. (refer to US Pat. No. 7,658,921). ) IgG heavy chains are numbered.
  • YTE mutant antibodies greatly extend the half-life of antibodies in serum compared to wild-type antibodies, such as eg, Dall'Acqua et al, J. Biol. Chem. 281:23514-24 (2006) and US Patent No. 7,083,784 .
  • a mouse can be immunized with human PCSK9 or a fragment thereof, and the obtained antibody can be renatured, purified, and subjected to amino acid sequencing by a conventional method.
  • the antigen-binding fragment can also be prepared by a conventional method.
  • the antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions in a non-human CDR region.
  • the human FR germline sequence can be obtained from the ImMunoGeneTics (IMGT) website http://imgt.cines.fr by comparing the IMGT human antibody variable region germline gene database and MOE software, or from the Immunoglobulin Journal, 2001 ISBN 014441351. obtain.
  • IMGT ImMunoGeneTics
  • the engineered antibodies or antigen-binding fragments of the invention can be prepared and purified by conventional methods.
  • a cDNA sequence encoding a heavy chain (SEQ ID NO: 28) and a light chain (SEQ ID NO: 30) can be cloned and recombined into a GS expression vector.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminal site of the Fc region.
  • Stable clones were obtained by expressing antibodies that specifically bind to human PCSK9. Positive clones were expanded in serum-free medium in a bioreactor to produce antibodies.
  • the culture medium from which the antibody is secreted can be purified by a conventional technique. For example, purification is carried out using an A or G Sepharose FF column containing an adjusted buffer. The non-specifically bound components are washed away. The bound antibody was eluted by a pH gradient method, and the antibody fragment was detected by SDS-PAGE and collected. The antibody can be concentrated by filtration in a conventional manner. Soluble mixtures and multimers can also be removed by conventional methods such as molecular sieves, ion exchange. The resulting product needs to be frozen immediately, such as -70 ° C, or lyophilized.
  • administering when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to an exogenous drug, therapeutic agent, diagnostic agent or composition and animal, human, subject Contact of the test subject, cell, tissue, organ or biological fluid.
  • administering can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods.
  • Treatment of the cells includes contact of the reagents with the cells, and contact of the reagents with the fluid, wherein the fluids are in contact with the cells.
  • administeristering and “treating” also means treating, for example, cells in vitro and ex vivo by reagents, diagnostics, binding compositions, or by another cell.
  • Treatment when applied to a human, veterinary or research subject, refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.
  • Treatment means administering to a patient a therapeutic agent for internal or external use, for example a composition comprising any of the binding compounds of the present invention, the patient having one or more symptoms of the disease, and the therapeutic agent is known to have Therapeutic effect.
  • a therapeutic agent is administered in a subject or population to be treated to effectively alleviate the symptoms of one or more diseases to induce such symptoms to degenerate or to inhibit the progression of such symptoms to any degree of clinical right measurement.
  • the amount of therapeutic agent also referred to as "therapeutically effective amount" effective to alleviate the symptoms of any particular disease can vary depending on a variety of factors, such as the patient's disease state, age and weight, and the ability of the drug to produce a desired effect in the patient.
  • Whether the symptoms of the disease have been alleviated can be assessed by any clinical test method commonly used by a physician or other professional health care provider to assess the severity or progression of the condition. While embodiments of the invention (e.g., methods of treatment or preparations) may be ineffective in ameliorating the symptoms of each target disease, any statistical test methods known in the art, such as Student's t-test, chi-square test, Mann's, and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that the target disease symptoms should be alleviated in a statistically significant number of patients.
  • any statistical test methods known in the art such as Student's t-test, chi-square test, Mann's, and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that the target disease symptoms should be alleviated in a statistically significant number of patients.
  • Constantly modified refers to amino acids in other amino acid substitution proteins having similar characteristics (eg, charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.), such that Changes are made without altering the biological activity of the protein. It will be appreciated by those skilled in the art that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., Page 224, (4th edition)). In addition, substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity.
  • an "effective amount” includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition.
  • An effective amount also means an amount sufficient to allow or facilitate the diagnosis.
  • An effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the overall health of the patient, the methodological route and dosage of the administration, and the severity of the side effects.
  • An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • Exogenous refers to a substance that is produced outside of a living being, cell or human, depending on the situation.
  • Endogenous refers to a substance produced in a cell, organism or human body, depending on the circumstances.
  • “Homology” refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When positions in both comparison sequences are occupied by the same base or amino acid monomer subunit, for example if each position of two DNA molecules is occupied by adenine, then the molecule is homologous at that position .
  • the percent homology between the two sequences is a function of the number of matches or homology positions shared by the two sequences divided by the number of positions compared x 100. For example, in the optimal alignment of sequences, if there are 6 matches or homologs in 10 positions in the two sequences, then the two sequences are 60% homologous. In general, comparisons are made when the maximum sequence of homology is obtained by aligning the two sequences.
  • the expression "cell”, “cell line” and “cell culture” are used interchangeably and all such names include progeny.
  • the words “transformants” and “transformed cells” include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that all offspring may not be exactly identical in terms of DNA content due to intentional or unintentional mutations. Mutant progeny having the same function or biological activity as screened for in the originally transformed cell are included. In the case of a different name, it is clearly visible from the context.
  • PCR polymerase chain reaction
  • oligonucleotide primers can be designed; these primers are identical or similar in sequence to the corresponding strand of the template to be amplified.
  • the 5' terminal nucleotides of the two primers may coincide with the ends of the material to be amplified.
  • PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA, phage or plasmid sequences transcribed from total cellular RNA, and the like. See, in general, Mullis et al. (1987) Cold Spring Harbor Symp. Ouant. Biol. 51:263; Editing by Erlich, (1989) PCR TECHNOLOGY (Stockton Press, N.Y.).
  • PCR used herein is considered as an example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, which comprises using a known nucleic acid and a nucleic acid polymerase as a primer to amplify or Produce a specific portion of the nucleic acid.
  • “Pharmaceutical composition” means a mixture comprising one or more compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, with other chemical components, such as physiological/pharmaceutically acceptable Carrier and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration of the organism, which facilitates the absorption of the active ingredient and thereby exerts biological activity.
  • the invention is further described in the following examples, which are not intended to limit the scope of the invention.
  • the experimental methods in the examples of the present invention which do not specify the specific conditions are usually in accordance with conventional conditions, such as the cold spring harbor antibody technology experiment manual, molecular cloning manual; or according to the conditions recommended by the raw material or commodity manufacturer.
  • Reagents without specific source are routine reagents purchased from the market.
  • the UniProt Proprotein convertase subtilisin/kexin type 9 (human PCSK9, Uniprot number: Q8MBP7) was used as a template for the PCSK9 of the present invention, and the amino acid sequences of the antigen and the detection protein of the present invention were designed, and the fusion was further based on the PCSK9 protein.
  • Labels such as his-tag or immuno-promoting peptides such as PADRE peptides were cloned into pTT5 vector (Biovector, Cat#: 102762) or pTargeT vector (promega, A1410), transiently expressed in 293 cells or stably expressed in CHO-S. Purification, obtaining the antigen encoding the present invention and the protein for detection.
  • PCSK9-His6 for immunogenic mice or detection reagents
  • the horizontal line is the signal peptide and the italic part is the His6-tag (6 histidine tag).
  • PCSK9 PCSK9-PADRE-His6 with PADRE peptide and His tag, as an immunogen, the PADRE peptide contained can promote immunity;
  • the horizontal line is the signal peptide
  • the double-line part is the linker
  • the dotted line is the PADRE peptide
  • the italic part is the His6-tag.
  • PCSK9 and his-tag fusion protein with PCV cleavage site PCSK9-TEV-His6, N-PCSK9 (N-terminal pCSK9 domain) can be obtained by TEV digestion as an immunogen;
  • the horizontal line is the signal peptide
  • the double-lined part is the TEV cleavage site
  • the italic part is the His6-tag.
  • PCSK9-D374Y mutant protein with his tag PCSK9-D374Y-His6 as a detection reagent
  • the horizontal line is the signal peptide and the italic part is the His6-tag.
  • PCSK9 PCSK9 protein inserted into biotin-accepting peptide BP15 and his tag: PCSK9-BP15-His6, as a detection reagent, the position of BP15 peptide can be biotinylated during expression, exempting in vitro biotin labeling and possible conformational changes;
  • the cross-hatched portion is the signal peptide
  • the double-lined portion is the biotin acceptor peptide
  • the italicized portion is the His6-tag.
  • PCSK9-Y PCSK9D374Y mutant protein inserted into biotin acceptor peptide and his tag: PCSK9-D374Y-BP15-His6, detection protein;
  • the cross-hatched portion is the signal peptide
  • the double-lined portion is the biotin acceptor peptide
  • the italicized portion is the His6-tag.
  • FcLR extracellular domain fragment of pCSK9 receptor protein with Flag tag and His tag LDLR-ECD-Flag-His6, detection reagent
  • the horizontal line is the signal peptide
  • the double-line part is the Flag label
  • the italic part is the His6-tag
  • LDLR-Fc a shortened form of the LDLR extracellular domain fragment and the hIgG1-Fc fusion protein (having binding activity to PCSK9): LDLR-sECD-Fc (hIgG1) as a detection reagent
  • the cross-hatched portion is a signal peptide
  • the double-scored portion is a shortened form of an LDLR extracellular domain fragment (LDLR-sECD) having a binding activity to PCSK9
  • the italicized portion is a hIgG1-Fc portion
  • the cross-hatched portion is the signal peptide
  • the double-lined portion is the more shortened form of the LDLR extracellular domain fragment (LDLR-ssECD) with PCSK9 binding activity
  • the italicized portion is the hIgG1-Fc portion.
  • the cell expression supernatant sample was centrifuged at high speed to remove impurities, and the buffer was exchanged for PBS, and imidazole was added to a final concentration of 5 mM.
  • the nickel column was equilibrated with PBS solution containing 5 mM imidazole and rinsed 2-5 column volumes.
  • the displaced supernatant sample was placed on an IMAC column.
  • the column was washed with PBS containing 5 mM imidazole until the A280 reading dropped to baseline.
  • the column was washed with PBS + 10 mM imidazole, the non-specifically bound heteroprotein was removed, and the effluent was collected.
  • the protein of interest was eluted with PBS containing 300 mM imidazole, and the eluted peak was collected.
  • the collected eluate was concentrated and further purified by gel chromatography Superdex 200 (GE), and the mobile phase was PBS. Depolymerized peaks were collected and the eluted peaks were collected.
  • the obtained protein was identified by electrophoresis, peptide mapping, LC-MS as correct and sub-equipment.
  • PCSK9-His6 His-tagged PCSK9-His6 (SEQ ID NO: 1), PCSK9-PADRE-His6 (SEQ ID NO: 2), PCSK9-TEV-His6 (SEQ ID NO: 3) PCSK9-D374Y-His6 (SEQ ID NO) : 4), PCSK9-BP15-His6 (SEQ ID NO: 5), PCSK9-D374Y-BP15-His6 (SEQ ID NO: 6) is used as an immunogen or detection reagent for the antibody of the present invention.
  • PCSK9-TEV-His6 was purified and digested by TEV enzyme, and the digested product was used to remove TEV enzyme, uncut intact PCSK9-TEV-His6 or excised His-tagged C-terminal domain fragment by IMAC column.
  • the IMAC effluent was concentrated to obtain a PCSK9 fragment (abbreviated as N-PCSK9) leaving only the N-terminal domain, and used as an immunogen for mouse immunization.
  • the sample was centrifuged at high speed to remove impurities and concentrated to an appropriate volume.
  • the flag affinity column was equilibrated with 0.5 x PBS and washed 2-5 column volumes.
  • the supernatant cells were subjected to supernatant analysis and the supernatant samples were applied to the column.
  • the column was rinsed with 0.5 x PBS until the A 280 reading dropped to baseline.
  • the column was washed with PBS containing 0.3 M NaCl, and the protein was washed and collected.
  • the protein of interest was eluted with 0.1 M acetic acid (pH 3.5-4.0) and collected to adjust the pH to neutral.
  • the collected eluate was concentrated and further purified by gel chromatography Superdex 200 (GE), and the mobile phase was PBS.
  • LDLR-ECD-Flag-His6 SEQ ID NO: 7 with FLAG/His6 tag was obtained for performance testing of the antibodies of the present invention.
  • the cell expression supernatant sample was centrifuged at high speed to remove impurities, and concentrated to an appropriate volume and applied to a Protein A column. Rinse the column with PBS until the A280 reading drops to baseline.
  • the protein of interest was eluted with 100 mM sodium acetate pH 3.0 and neutralized with 1 M Tris-HCl. The eluted sample was appropriately concentrated and further purified by PBS-balanced gel chromatography Superdex 200 (GE). The peak of the depolymerized product was collected and used. This method was used to purify LDLR-sECD-Fc (hIgG1) (SEQ ID NO: 8) and LDLR-ssECD-Fc (hIgG1) (SEQ ID NO: 9). Both can be used as PCSK9 antibody functional tests.
  • Anti-human PCSK9 monoclonal antibodies are produced by immunizing mice.
  • Experimental SJL white mice female, 6 weeks old (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., animal production license number: SCXK (Beijing) 2012-0001).
  • Feeding environment SPF level. After the mice were purchased, the laboratory environment was kept for 1 week, 12/12 hours light/dark cycle adjustment, temperature 20-25 ° C; humidity 40-60%. Mice that have adapted to the environment are immunized (A/B) in two regimens, 6-10 per group.
  • the immunizing antigen is His-tagged human PCSK9-His6 (SEQ ID NO: 1), PCSK9-PADRE-His6 (SEQ ID NO: 2), and N-PCSK9 (SEQ ID NO: 3).
  • Protocol A was emulsified with Freund's adjuvant (sigma Lot Num: F5881/F5506): the first use of Freund's complete adjuvant (CFA), and the rest of the booster with Freund's incomplete adjuvant (IFA).
  • the ratio of antigen to adjuvant was 1:1, 100 ⁇ g/only (first aid), 50 ⁇ g/only (boost boost).
  • Scheme B was cross-immunized with Titermax (sigma Lot Num: T2684) and Alum (Thremo Lot Num: 77161).
  • the ratio of antigen to adjuvant (titermax) was 1:1, and the ratio of antigen to adjuvant (Alum) was 3:1, 10-20 ⁇ g/only (first exempt), and 5 ⁇ g/only (boosting).
  • mice in the serum were selected to have high antibody titers (see Test Methods 1 and 2 below, combined with the ELISA method of PCSK9) and spleen cell fusion was performed in mice with titer-to-platform, and the selected mice were spurted 72 hours before the fusion, PCSK9- His6 10 ⁇ g/only, intraperitoneal injection.
  • Spleen lymphocytes and myeloma cell Sp2/0 cells were optimized using an optimized PEG-mediated fusion step ( CRL-8287 (TM ) was fused to obtain hybridoma cells.
  • HAT complete medium RPMI-1640 medium containing 20% FBS, 1 ⁇ HAT and 1 ⁇ OPI
  • RPMI-1640 medium containing 20% FBS, 1 ⁇ HAT and 1 ⁇ OPI
  • 96-well cell culture plates (1 ⁇ 10 5 /150 ⁇ l). /well)
  • HAT complete medium was added, 50 ⁇ l/well, and incubated at 37 ° C, 5% CO 2 .
  • the whole medium was changed, and the medium was HT complete medium (RPMI-1640 medium containing 20% FBS, 1 ⁇ HT and 1 ⁇ OPI), 200 ⁇ l/well, Incubate at 37 ° C, 5% CO 2 .
  • HT complete medium RPMI-1640 medium containing 20% FBS, 1 ⁇ HT and 1 ⁇ OPI
  • ELISA assays in combination with PCSK9 or PCSK9-Y were performed according to cell growth density (see Test Examples 1 and 2).
  • the positive well cells combined with ELISA were subjected to a blocking ELISA assay for binding of PCSK9 or PCSK9-Y to LDLR (see Test Examples 3 and 4), and the positive wells were exchanged and expanded into 24-well plates according to cell density.
  • the cell line transferred into the 24-well plate was subjected to retesting and then subjected to seed conservation and first subcloning.
  • the first subcloning screen (see Test Examples 1 and 2) was positive for conservation and a second subcloning.
  • the second subcloning was positive (see Test Examples 1 and 2) for conservation and protein expression. Multiple fusions were obtained to obtain hybridoma cells that blocked the binding of PCSK9 or PCSK9-Y to LDLR (see Test Examples 3 and 4).
  • the hybridoma clone mAb-001 was screened by blocking assay and binding assay, and the antibody was further prepared by serum-free cell culture, and the antibody was purified according to the purification example for use in the test example.
  • the murine anti-variable region sequences of the hybridoma clone mAb-001 were determined as follows:
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the italicized FR sequence in the sequence, underlined as the CDR sequence.
  • the two murine antibodies were selected by aligning the IMGT human antibody heavy light chain variable region germline gene database and MOE software, respectively, to select the heavy and light chain variable region germline gene with high homology to mAb-001 as a template.
  • the CDRs were each transplanted into the corresponding human template to form a variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the amino acid residues are determined and annotated by the Kabat numbering system.
  • the humanized light chain template of the murine antibody mAb-001 was IGKV1-39*01 and hjk2.1, and the humanized heavy chain template was IGHV1-2*02 and hjh2. After humanization, the humanized antibody h001- was obtained.
  • the sequence of the variable region of 1 is as follows:
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the italicized FR sequence in the sequence, underlined as the CDR sequence.
  • variable region of the humanized antibody h001-1 consists of the light chain h001_VL1 and the heavy chain h001_VH.1A. Other analogies.
  • the PCSK9 antibody obtained by the present invention has high binding activity to PCSK9 and PCSK9-Y; and can effectively block the binding between PCSK9/PCSK9-Y and LDLR.
  • the method for constructing and expressing an anti-human PCSK9 humanized antibody of the present invention is as follows:
  • Primer design use the online software DNAWorks(v3.2.2) (http://helixweb.nih.gov/dnaworks/) to design multiple primers to synthesize VH/VK gene fragments containing recombinants: 5'-30bp Signal peptide+ VH/VK+30bp CH1/CL-3'.
  • Primer design principle The target gene 2 is different from the target gene 1 by 2 aa, and the primer of the mutation site is also set, as shown in Fig. 1.
  • the expression vector pHr (with signal peptide and constant region gene (CH1-FC/CL) fragment) was designed using some special restriction enzymes, such as BsmBI, to distinguish the recognition sequence from the restriction site, as shown in Figure 2. Show. The vector was digested with BsmBI, and the gel was recovered for use.
  • BsmBI signal peptide and constant region gene
  • VH/VK contains the gene fragment required for recombination and BsmBI digestion and expression vector pHr (with signal peptide and constant region gene (CH1-FC/CL) fragment) was added to DH5 ⁇ competent cells in a ratio of 3:1, ice at 0 °C Bath for 30 min, heat shock at 42 ° C for 90 seconds, add 5 volumes of LB medium, incubate for 45 min at 37 ° C, coat LB-Amp plate, incubate overnight at 37 ° C, pick monoclonal and send to sequencing to obtain each clone.
  • pHr signal peptide and constant region gene
  • the antibodies of the invention may be, but are not limited to, the above design constructs.
  • the antibody and its mutant design were carried out by taking h001-4 as an example to obtain the IgG1 form of 1h001-4-WT: h001-4, that is, the humanized sequence combination h001-4, which binds to the heavy chain constant region derived from human IgG1, and The light chain constant region of the human kappa chain; the 2h001-4-YTE: h001-4-IgG1-YTE form, ie the humanized sequence combination h001-4, binds to the heavy chain constant region of the mutated human IgG1 (YTE mutation), A light chain constant region from a human kappa chain.
  • the mutated human IgG1 may also be a mutation of another form.
  • the obtained antibody and the mutated antibody were tested for affinity using BIAcore (Test Example 6), and the results are shown in Table 9.
  • the sequence of the humanized PCSK9 humanized antibody (IgG1 and IgG1-YTE forms) constructed and expressed in the present invention is as follows:
  • the heavy chain constant region is derived from human IgG1 and the light chain constant region is derived from the human kappa light chain:
  • Heavy chain amino acid sequence (human IgG1):
  • h001-4-IgG1-YTE (light chain is h001-4-kappa: SEQ ID NO: 30)
  • Heavy chain amino acid sequence IgG1-YTE
  • Test Example 1 ELISA assay of PCSK9 antibody binding to wild-type PCSK9 protein
  • the binding test of the PCSK9 antibody of the present invention to PCSK9 was detected by the amount of binding of the antibody to wild type PCSK9 (WT-PCSK9, SEQ ID NO: 5) immobilized on an ELISA plate.
  • Streptavidin (sigma, CAT #S4762) was diluted to 2 ⁇ g/ml with PBS, coated on a 96-well ELISA plate, and placed at 4 ° C overnight. After washing, the cells were blocked with Tris buffer (containing 0.9 mM calcium chloride, 0.05% Tween 20 and 5% skim milk powder) for 2 hours at 37 °C. Wash the plate and add internally produced biotin-labeled PCSK9 (bio-WT-PCSK9, with Tris containing 0.9 mM calcium chloride, 0.05% Tween 20 and 1% skim milk powder) Dilute with buffer) 100 ⁇ l/well and incubate for 1 hour at 37 °C.
  • the plate was washed, and anti-PCSK9 antibody samples diluted in different concentrations were added and incubated at 37 ° C for 1 hour.
  • the plate was washed again, and horseradish peroxidase-goat anti-human (H+L) antibody (jackson, CAT#109-035-088) was added and incubated at 37 ° C for 1 hour.
  • the plate was washed again and added to a tetramethylbenzidine solution for color development. Finally, the stop solution was added, the OD450 was measured on a microplate reader, and its EC50 value was calculated.
  • the binding ELISA assay of the chimeric antibody of the present invention and the antibody after back-mutation with PCSK9 is shown in Table 5.
  • the binding test of the PCSK9 antibody of the present invention to PCSK9-Y was detected by the amount of binding of the antibody to PCSK9-Y (mutant PCSK9, SEQ ID NO: 6) immobilized on an ELISA plate.
  • Streptavidin (sigma, CAT #S4762) was diluted to 2 ⁇ g/ml with PBS, coated on a 96-well ELISA plate, and placed at 4 ° C overnight. After washing, the cells were blocked with Tris buffer (containing 0.9 mM calcium chloride, 0.05% Tween 20 and 5% skim milk powder) for 2 hours at 37 °C. Wash the plate and add internally produced biotinylated PCSK9-Y (bio-PCSK9-Y, diluted with Tris buffer containing 0.9 mM calcium chloride, 0.05% Tween 20 and 1% skim milk powder) 100 ⁇ l/well, 37 °C Incubate for 1 hour.
  • the plate was washed, and anti-PCSK9 antibody samples diluted in different concentrations were added and incubated at 37 ° C for 1 hour.
  • the plate was washed again, and horseradish peroxidase-goat anti-human (H+L) antibody (jackson, CAT#109-035-088) was added and incubated at 37 ° C for 1 hour.
  • the plate was washed again and added to a tetramethylbenzidine solution for color development. Finally, the stop solution was added, the OD450 was measured on a microplate reader, and its EC50 value was calculated.
  • the binding ELISA assay of the chimeric antibody, the back-mutated antibody and the mutant PCSK9 of the present invention is shown in Table 6.
  • Test Example 3 Blocking of LDLR-FC/PCSK9-Y binding by PCSK9 antibody
  • LDLR-FC was diluted with phosphate buffer to 2 ⁇ g/ml, coated on a 96-well ELISA plate (Costar, CAT #3590) and allowed to stand overnight at 4 °C. After washing, the cells were blocked with Tris buffer (containing 0.9 mM calcium chloride, 0.05% Tween 20 and 5% skim milk powder) for 2 hours at 37 °C. Wash the plate, add biotin-labeled PCSK9-Y (bio-PCSK9-Y, diluted to a final concentration of 1 ⁇ g/ml with Tris buffer containing 0.9 mM calcium chloride, 0.05% Tween 20 and 1% skim milk powder), and antibody.
  • Tris buffer containing 0.9 mM calcium chloride, 0.05% Tween 20 and 5% skim milk powder
  • a mixture of the sample (diluted with Tris buffer containing 0.9 mM calcium chloride, 0.05% Tween 20 and 1% skim milk powder) was incubated at 37 ° C for 1 hour. The plate was washed, and horseradish peroxidase-streptavidin (sigma, CAT #S2438) was added and incubated at 37 ° C for 1 hour. The plate was washed again and added to a tetramethylbenzidine solution for color development. Finally, the stop solution was added, the OD450 was measured on a microplate reader, and its IC50 value was calculated.
  • PCSK9 antibody of the invention is tested for binding to other forms of LDLR-FC (internally produced, sequence SEQ ID NO: 7 or SEQ ID NO: 9) and PCSK9-Y (SEQ ID NO: 5) using the methods described above. Blocking ability, experiments demonstrated that the PCSK9 antibody of the present invention can effectively block the binding between PCSK9 and the shortened form of LDLR.
  • Test Example 4 Blocking of LDLR-FC/PCSK9 binding by PCSK9 antibody
  • PCSK9 antibody of the invention is for LDLR-FC (internal production, sequence is SEQ ID NO: 8) and PCSK9 (SEQ ID NO: 5)
  • the blocking ability test of binding was determined by measuring the amount of binding of PCSK9 to LDLR in the presence of antibodies.
  • LDLR-FC was diluted to 5 ⁇ g/ml with phosphate buffer, coated on a 96-well ELISA plate and allowed to stand overnight at 4 °C. After washing, the cells were blocked with Tris buffer (containing 0.9 mM calcium chloride, 0.05% Tween 20 and 5% skim milk powder) for 2 hours at 37 °C.
  • biotin-labeled PCSK9 bio-WT-PCSK9, diluted to a final concentration of 2 ⁇ g/ml with Tris buffer containing 0.9 mM calcium chloride, 0.05% Tween 20 and 1% skim milk powder
  • antibody samples use A mixture of 100 ⁇ l/well of a mixture containing 0.9 mM calcium chloride, 0.05% Tween 20 and 1% skim milk powder diluted in Tris buffer was incubated at 37 ° C for 1 hour. The plate was washed, and horseradish peroxidase-streptavidin (sigma, CAT #S2438) was added and incubated at 37 ° C for 1 hour. The plate was washed again and added to a tetramethylbenzidine solution for color development. Finally, the stop solution was added, the OD450 was measured on a microplate reader, and its IC50 value was calculated.
  • HepG2 cells (Chinese Academy of Sciences Cell Bank, #CAT, TCHu72) were cultured in DMEM medium (Hyclone, #CAT SH30243.01B) (containing 10% fetal bovine serum, Gibco, #CAT 10099-141). When the cells covered 80-90%, the cells were counted at a rate of 1.5*10 4 cells/well after digestion and blown in a 96-well plate. After 24 hours, the medium was changed to DMEM, 10% non-lipoprotein serum (Millipore, CAT #LP4).
  • Human Fab capture molecules were covalently coupled to a CM5 biosensor chip (Cat. #BR-1000-12, according to the method described in the Human Fab Capture Kit (Cat. #28-9583-25, GE) instructions. GE), thereby affinity-captured the antibody to be tested, and then flowed through the human PCSK9 antigen (His-tagged human PCSK9: PCSK9-His6, SEQ ID NO: 1) on the surface of the chip, and the reaction signal was detected in real time using Biacore instrument to obtain binding. And the dissociation curve, the affinity value is obtained by fitting, see Table 9. After each cycle of dissociation was completed in the experiment, the biochip was washed and regenerated using the regeneration solution disposed in the human Fab capture kit (GE).
  • the PCSK9 antibody of the present invention has a strong affinity for human PCSK9 antigen.
  • PCSK9-Y The affinity of the PCSK9 antibody of the present invention to PCSK9-Y (SEQ ID NO: 4) was examined by a similar method as above, and it was revealed that the PCSK9 antibody of the present invention has a strong affinity with the PCSK9-Y antigen.
  • mice purchased from Shanghai Xipuer Bikai Experimental Animal Co., Ltd.
  • the internally produced human IgG and h001-4-WT antibody were administered at a dose of 10 mg/kg (human IgG, h001-4-WT antibody was prepared in PBS). The concentration is 1 mg/ml). Fasting was performed for 6 hours before blood was taken, blood was taken from the eyelids at 24, 48, 72, and 96 hours after administration, and left at 37 ° C for 1 hour, centrifuged at 3500 rpm for 10 minutes, and serum was stored at -80 ° C.
  • Serum LDL-c concentrations were measured using HDL and LDL/VLDL Cholesterol Quantification Kit and operated according to the kit instructions.
  • the concentration of serum LDL-c in normal mice is about 12 mg/dl. After injection of AAV8-PCSK9 virus, the concentration of LDL-c in the serum reached an average of 40 mg/dl. After grouping, after 24 hours of administration, the concentration of LDL-c in the h001-4-WT group decreased by 50% compared with the human IgG group; after 48 hours of administration, the concentration of LDL-c in the h001-4-WT group decreased 49.
  • h001-4-WT was able to reduce the concentration of LDL-c in the serum of mice overexpressing human PCSK9, and the efficacy lasted for 72 hours.
  • PK blood collection point is before administration, 15 minutes, 30 minutes, 1 hour, 3 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72, 96, 120 hours, 144 hours, 168 after administration. Hours, 336 hours, 504 hours, 672 hours.
  • the serum samples of the blood samples were tested for the content of h001-4-WT and h001-4-YTE by ELISA.
  • the method was as described in Test Example 1.
  • the results showed that the half-life of h001-4-WT in cynomolgus monkeys was 4 days. While h001-4-YTE has a half-life of 7.3 days in cynomolgus monkeys, YTE has a significantly longer in vivo half-life than WT.

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Abstract

提供一种PCSK9抗体，其抗原结合片段及其医药用途。提供包含所述PCSK9抗体CDR区的嵌合抗体、人源化抗体以及包含PCSK9抗体及其抗原结合片段的药物组合物，以及其作为降血脂药物的用途。特别地，涉及一种人源化的PCSK9抗体在制备用于治疗PCSK9介导的疾病或病症的药物中的用途。

Description

PCSK9抗体、其抗原结合片段及其医药用途 技术领域
本发明涉及PCSK9抗体、PCSK9抗体的抗原结合片段、包含所述PCSK9抗体CDR区的嵌合抗体、人源化抗体，以及包含所述PCSK9抗体及其抗原结合片段的药物组合物，以及其作为降血脂药物的用途。
背景技术
高胆固醇血症是一种以血清胆固醇水平升高为主要特征的脂类代谢异常疾病，其主要表现为血清胆固醇水平升高，导致胆固醇在血管聚集，形成动脉粥样硬化。大量的临床及实验研究结果都证实，脂质代谢异常和冠心病的发生、发展有着密切的关系。因此，降低血液中的胆固醇浓度成为了目前治疗和预防动脉粥样硬化的一个主要手段。
随着我国国民生活水平的快速提高，血脂异常也成为了危害我国城乡居民的主要因素。据2012年统计数据，我国每年死亡人数中约有40％死于心血管疾病。目前，我国成人血脂异常患病率为18.6％，估计全国血脂异常现患人数1.6亿。不同类型的血脂异常现患率分别为：高胆固醇血症2.9％，高甘油三酯血症11.9％，低高密度脂蛋白血症7.4％；另有3.9％的人血胆固醇边缘升高。2012年卫生部疾病预防控制专家委员会慢性病防治分委会达成的“慢性病防治中国专家共识”中提到，我国有3300万高胆固醇血症患者，而从局部地区看，我国血脂异常发病率情况远比上述数据要严重。
目前，临床上调脂药物主要以他汀类为主。立普妥(Liptor)作为全世界应用最广泛的降胆固醇药物，也是医药史上最畅销药物，通过阻断肝脏生产胆固醇的酶作用，减少胆固醇的生产，从而增加肝脏从血液中摄取更多的胆固醇，进而减低血液中胆固醇浓度。但是立普妥也有其不足之处，首先从数据看,立普妥可以降低30％-40％的低密度脂蛋白,但仍然有很多病人依然无法到达有效的降低血脂(低密度脂蛋白浓度<50mg/dL)，其次病人对立普妥的响应率也有人种差异。这些原因，致使病人需要一个更为有效的降低血脂的药物。
家族性高胆固醇血症(Familial hypercholesterolemia,FM)是一种常染色体单基因显性遗传性疾病，其临床特征为血总胆固醇和低密度脂蛋白胆固醇(low density lipoprotein-cholesterol,LDL-c)显著升高、睑黄瘤、角膜弓以及早发的心血管疾病。低密度脂蛋白受体(LDL receptor,LDLR)基因突变致其缺陷或缺乏，LDL-c不能顺利转运到肝脏清除，以致血中LDL-c水平升高。目前已明确3种基因与FM的发生有关，它们分别是LDLR基因、载脂蛋白B100基因和PCSK9(proprotein convertase subtilisin/kexin type 9)基因。
前蛋白转化酶枯草溶菌素9(PCSK9)是一种前蛋白转化酶，属于分泌的枯草杆菌酶家族的蛋白酶K亚族。该编码蛋白是作为可溶性酶原合成的，在内质网中经过自身催化分子内加工。实验结果显示，PCSK9促进LDL受体的降解从而增加血浆中LDL胆固醇含量，而LDL受体介导肝内的LDL胞吞过程，后者是从循环系统清除LDL的主要途径。研究发现，12.5％的高胆固醇血症(ADH)患者检测有PCSK9基因突变。PCSK9突变形式多样，根据突变对PCSK9调节LDL-C水平的不同影响可分为两类：功能缺失型和功能获得型。其中功能缺失型突变与低血胆固醇水平有关，有预防冠状动脉粥样硬化性心脏病发生的作用，非洲人群中低胆固醇的PCSK9突变率高于其他种族。PCSK9功能获得型突变体通过增加PCSK9的功能、降低LDLR的表达而升高血浆胆固醇水平，可以导致严重高胆固醇血症和早发冠状动脉粥样硬化性心脏病，目前发现的PCSK9功能获得型突变包括：D374Y、S127R、F216L、N157K、R306S等。其中，与PCSK9野生型相比，D374Y突变体细胞表面的LDLR减少了36％，S127R突变有相应减少了10％。
目前PCSK9作为一个极具潜力的，新的靶标已成为高胆固醇血症研究的热点，对于深入了解胆固醇代谢的机制和寻求新的治疗手段有重要意义。有多家跨国制药公司在研发针对PCSK9的单克隆抗体，它通过在血液中中和PCSK9，从而增加了肝脏表面LDL受体的浓度，进而达到降低血液中LDL浓度的目的。相关的专利有WO2011111007、WO2011072263、WO2013170367、WO2013169886、WO2013148284、WO2013091103、WO2013039958、WO2013039969、WO2013016648、WO2013008185、WO2012170607、WO2012168491、WO2012154999、WO2012109530、WO2012101251、WO2012088313、US8829165B2、US8563698B2、US8859741B2、US8871913B2、US8871914B2、US8883983B2、WO2012058137和WO2012054438。
本发明提供有着更高亲和力、更高选择性、更高生物活性的PCSK9抗体。
发明内容
本发明提供一种PCSK9抗体或其抗原结合片段，其包含一个或多个选自以下的CDR：如序列SEQ ID NO:12、SEQ ID NO:13或SEQ ID NO:14所示的、或与SEQ ID NO:12、SEQ ID NO:13或SEQ ID NO:14具有至少95％序列同一性的序列所示的HCDR；和如序列SEQ ID NO:15、SEQ ID NO:16或SEQ ID NO:17所示的、或与SEQ ID NO:15、SEQ ID NO:16或SEQ ID NO:17具有至少95％序列同一性的序列所示的LCDR。
在本发明另一个优选的实施方案中，本发明所述的PCSK9抗体或其抗原结合片段，其包含分别如SEQ ID NO:12、SEQ ID NO:13和SEQ ID NO:14所示的HCDR1、HCDR2和HCDR3，或包含分别与SEQ ID NO:12、SEQ ID NO:13和SEQ ID NO:14具有至少95％序列同一性的序列所示的HCDR1、HCDR2和 HCDR3。
在本发明另一个优选的实施方案中，本发明所述的PCSK9抗体或其抗原结合片段，其包含分别如SEQ ID NO:15、SEQ ID NO:16和SEQ ID NO:17所示的LCDR1、LCDR2和LCDR3，或包含分别与SEQ ID NO:15、SEQ ID NO:16和SEQ ID NO:17具有至少95％序列同一性的序列所示的LCDR1、LCDR2和LCDR3。
上述的具有至少95％序列同一性的氨基酸序列，可以是通过亲和力成熟的技术手段，对本发明的CDR区进行突变获得的。
在本发明另一个优选的实施方案中，本发明所述的PCSK9抗体或其抗原结合片段，其中所述的抗体或其抗原结合片段为鼠源抗体或其片段。
在本发明另一个优选的实施方案中，本发明所述的PCSK9抗体或其抗原结合片段，其中所述PCSK9抗体的轻链可变区进一步包含鼠源κ链或鼠源κ链变体的轻链FR区、或者鼠源λ链或鼠源λ链变体的轻链FR区；其中所述PCSK9抗体的重链可变区进一步包含鼠源IgG1或其变体的重链FR区、或IgG2或其变体的重链FR区、或IgG3或其变体的重链FR区。
在本发明另一个优选的实施方案中，本发明所述的PCSK9抗体或其抗原结合片段，其中所述的鼠源抗体包含SEQ ID NO:10的重链可变区序列和SEQ ID NO:11的轻链可变区序列。
在本发明另一个优选的实施方案中，本发明所述的PCSK9抗体或其抗原结合片段，其中所述PCSK9抗体的轻链进一步包含鼠源κ链或其变体的轻链恒定区、或者鼠源λ链或其变体的轻链恒定区；其中所述PCSK9抗体重链进一步包含鼠源IgG1或其变体的重链恒定区、或IgG2或其变体的重链恒定区、或IgG3或其变体的重链恒定区。
在本发明另一个优选的实施方案中，本发明所述的PCSK9抗体或其抗原结合片段，其中所述的抗体或其抗原结合片段为嵌合抗体或其片段。
在本发明另一个优选的实施方案中，本发明所述的PCSK9抗体或其抗原结合片段，其中所述的抗体或其抗原结合片段为人源化抗体或其片段。
在本发明另一个优选的实施方案中，本发明所述的PCSK9抗体或其抗原结合片段，其中所述的人源化抗体重链可变区上的重链FR区序列来源于人种系重链，IGHV1-2*02和hjh2的组合序列及其突变序列；其包含人种系重链IGHV1-2*02的FR1、FR2、FR3区和hjh2的FR4区及其突变序列，或与其具有至少95％序列同一性的氨基酸序列。
在本发明另一个优选的实施方案中，本发明所述的PCSK9抗体或其抗原结合片段，其中所述的人源化抗体含有SEQ ID NO:18所示的重链可变区；或SEQ ID NO:18变体所示的重链可变区；其中所述SEQ ID NO:18变体是在SEQ ID NO:18所示的重链可变区位置上具有0-10个氨基酸变化的序列。所述的氨基酸变化可以是用现有技术，为提高抗体如亲和性、半衰期等性能做的改进，如用亲和力成熟 修改CDR区的氨基酸，或者用回复突变修改FR区的氨基酸。
在本发明另一个优选的实施方案中，本发明所述的PCSK9抗体或其抗原结合片段，其中所述的人源化抗体重链FR区序列有0-10个氨基酸的回复突变，优选为一个或多个选自T30N,R87T,R72A,T74K,M48I,V68A,M70L,R38K和R67K的氨基酸回复突变。
在本发明另一个优选的实施方案中，本发明所述的PCSK9抗体或其抗原结合片段，其中所述人源化抗体包含选自SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22和SEQ ID NO:23的序列所示的重链可变区。
在本发明另一个优选的实施方案中，本发明所述的PCSK9抗体或其抗原结合片段，其中所述的人源化抗体轻链可变区上的轻链FR区序列来源于人种系轻链模板IGKV1-39*01和hjk2.1的组合序列及其突变序列；其包含人种系轻链IGKV1-39*01的FR1、FR2、FR3区和hjk2.1的FR4区及其突变序列，或与其具有至少95％序列同一性的氨基酸序列。
在本发明另一个优选的实施方案中，本发明所述的PCSK9抗体或其抗原结合片段，其中所述人源化抗体进一步包含SEQ ID NO:24所示的、或SEQ ID NO:24变体所示的轻链可变区；所述的SEQ ID NO:24变体是在SEQ ID NO:24所示的轻链可变区位置上具有0-10的氨基酸变化。所述的氨基酸变化可以是用现有技术，为提高抗体如亲和性、半衰期等性能做的改进，如用亲和力成熟修改CDR区的氨基酸，或者用回复突变修改FR区的氨基酸。
在本发明另一个优选的实施方案中，本发明所述的PCSK9抗体或其抗原结合片段，其中所述的SEQ ID NO:24变体是在SEQ ID NO:24所示的轻链可变区的FR区位置上具有0-10个氨基酸的回复突变；优选的，所述回复突变选自T5S,S66D,Q3V和A49S的氨基酸回复突变；优选为A43S的氨基酸变化。
在本发明另一个优选的实施方案中，本发明所述的PCSK9抗体或其抗原结合片段，其中所述人源化抗体包含选自SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27的序列所示的轻链可变区。
在本发明另一个优选的实施方案中，本发明所述的PCSK9抗体或其抗原结合片段，其中所述人源化抗体包含重链可变区序列和/或轻链可变区序列，所述重链可变区序列选自SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22和SEQ ID NO:23所示的序列，或所述重链可变区序列选自与SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22和SEQ ID NO:23具有至少95％序列同一性的序列；所述轻链可变区序列选自SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27所示的序列，或所述轻链可变区序列选自选自与SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27具有至少95％序列同一性序列。
在本发明另一个优选的实施方案中，本发明所述的PCSK9抗体或其抗原结合片段，其中所述的PCSK9抗体包含选自以下的重链可变区和轻链可变区：
1)SEQ ID NO:18的重链可变区和SEQ ID NO:25的轻链可变区，
2)SEQ ID NO:18的重链可变区和SEQ ID NO:26的轻链可变区，
3)SEQ ID NO:18的重链可变区和SEQ ID NO:27的轻链可变区，
4)SEQ ID NO:19的重链可变区和SEQ ID NO:24的轻链可变区，
5)SEQ ID NO:19的重链可变区和SEQ ID NO:25的轻链可变区，
6)SEQ ID NO:19的重链可变区和SEQ ID NO:26的轻链可变区，
7)SEQ ID NO:19的重链可变区和SEQ ID NO:27的轻链可变区，
8)SEQ ID NO:20的重链可变区和SEQ ID NO:24的轻链可变区，
9)SEQ ID NO:20的重链可变区和SEQ ID NO:25的轻链可变区，
10)SEQ ID NO:20的重链可变区和SEQ ID NO:26的轻链可变区，
11)SEQ ID NO:20的重链可变区和SEQ ID NO:27的轻链可变区，
12)SEQ ID NO:21的重链可变区和SEQ ID NO:24的轻链可变区，
13)SEQ ID NO:21的重链可变区和SEQ ID NO:25的轻链可变区，
14)SEQ ID NO:21的重链可变区和SEQ ID NO:26的轻链可变区，
15)SEQ ID NO:21的重链可变区和SEQ ID NO:27的轻链可变区，
16)SEQ ID NO:22的重链可变区和SEQ ID NO:24的轻链可变区，
17)SEQ ID NO:22的重链可变区和SEQ ID NO:25的轻链可变区，
18)SEQ ID NO:22的重链可变区和SEQ ID NO:26的轻链可变区，
19)SEQ ID NO:22的重链可变区和SEQ ID NO:27的轻链可变区，
20)SEQ ID NO:23的重链可变区和SEQ ID NO:24的轻链可变区，
21)SEQ ID NO:23的重链可变区和SEQ ID NO:25的轻链可变区，
22)SEQ ID NO:23的重链可变区和SEQ ID NO:26的轻链可变区，
23)SEQ ID NO:23的重链可变区和SEQ ID NO:27的轻链可变区，和
24)SEQ ID NO:18的重链可变区和SEQ ID NO:24的轻链可变区。
在本发明另一个优选的实施方案中，本发明所述的PCSK9抗体或其抗原结合片段，其中所述PCSK9抗体的重链进一步包含人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，或与其具有至少95％序列同一性的氨基酸序列；优选包含人源IgG1、IgG2或IgG4或使用氨基酸突变延长抗体在血清中的半衰期的IgG1、IgG2或IgG4变体的重链恒定区，更优选包含引入YTE突变的IgG1、IgG2或IgG4重链恒定区；
其中所述PCSK9抗体的轻链进一步包含人源κ、λ链或其变体的恒定区，或与其具有至少95％序列同一性的氨基酸序列。
在本发明另一个优选的实施方案中，本发明所述的PCSK9抗体或其抗原结合片段，其中所述人源化抗体包含选自以下的重链和轻链：
1)SEQ ID NO:28的重链和SEQ ID NO:30的轻链，和
2)SEQ ID NO:32的重链和SEQ ID NO:30的轻链。
本发明进一步提供一种药物组合物，其含有治疗有效量的如上所述的PCSK9抗体或其抗原结合片段，以及一种或多种药学上可接受的载体、稀释剂或赋形剂。
本发明进一步提供一种编码如上所述的PCSK9抗体或其抗原结合片段的DNA分子。
本发明进一步提供一种如上所述的DNA分子的表达载体。
本发明进一步提供一种如上所述的表达载体转化的宿主细胞，所述宿主细胞选自原核细胞和真核细胞，优选为真核细胞，更优选哺乳动物细胞。
本发明进一步提供一种如上所述的PCSK9抗体或其抗原结合片段或如上所述的药物组合物，在制备用于治疗PCSK9介导的疾病或病症的药物中的用途，其中所述的疾病或病症优选为胆固醇相关疾病(其包括“血清胆固醇相关疾病”)；更优选为高胆固醇血症、心脏病、代谢综合征、糖尿病、冠状动脉心脏病、卒中、心血管疾病、阿尔茨海默病和一般性的异常脂血症；最优选高胆固醇血症、异常脂血症、动脉粥样硬化、CVD或冠状动脉心脏病。
可以使用本发明的抗体诊断的示例性疾病包括胆固醇相关疾病(其包括“血清胆固醇相关疾病”)，其包括以下的任何一种或多种：高胆固醇血症、心脏病、代谢综合征、糖尿病、冠状动脉心脏病、卒中、心血管疾病、阿尔茨海默病和一般性的异常脂血症(其显示为例如提高的总血清胆固醇、提高的LDL、提高的甘油三酯、提高的极低密度脂蛋白(VLDL)和/或低的HDL)。
在一方面中，本发明提供治疗或预防个体中的高胆固醇血症和/或至少一种以下症状的方法：异常脂血症、动脉粥样硬化、心血管疾病(CVD)或冠状动脉心脏病，所述方法包括向所述个体施用有效量的抗PCSK9抗体。本发明还提供有效量的拮抗胞外或循环PCSK9的抗PCSK9抗体在制备药物中的用途，所述药物用于治疗或预防个体的高胆固醇血症和/或至少一种以下症状：异常脂血症、动脉粥样硬化、CVD或冠状动脉心脏病。
附图说明
图1:本发明抗体载体构建中的引物设计示意图。
图2:本发明抗体载体构建示意图。
图3:不同h001-4-YTE抗PCSK9抗体浓度中HepG2细胞的LDL摄取变化。数据结果显示PCSK9抗体能够促进HepG2细胞摄取LDL。
图4:不同h001-4-WT抗PCSK9抗体浓度中HepG2细胞的LDL摄取变化。数据结果显示PCSK9抗体能够促进HepG2细胞摄取LDL。
图5:注射h001-4-WT抗PCSK9抗体的小鼠血清中LDL-c浓度随时间变化(*：p<0.05,vs IgG，**：p<0.01,vs IgG)。数据结果显示PCSK9抗体能够降低过表达人PCSK9的小鼠血清中LDL-c浓度。
图6:注射h001-4-WT抗PCSK9抗体的小鼠血清中相对IgG组的LDL-c浓度变化。数据结果显示相对IgG组，PCSK9抗体能够降低过表达人PCSK9的小鼠血清中LDL-c浓度。
图7:本发明抗体在食蟹猴体内药效及药代检测。附图显示h001-4-WT和h001-4-YTE均能够明显降低食蟹猴体内LDL的含量，且h001-4-YTE的降低持续时间要优于h001-4-WT。
具体实施方式
术语定义
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。
本发明所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中所述。
本发明所述的“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链、和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。
在本发明中，本发明所述的抗体轻链可变区可进一步包含轻链恒定区，所述的轻链恒定区包含人源或鼠源的κ、λ链或其变体。
在本发明中，本发明所述的抗体重链可变区可进一步包含重链恒定区，所述的重链恒定区包含人源或鼠源的IgG1、IgG2、IgG3、IgG4或其变体。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(Fv区)；靠近C端的其余氨基酸序列相对稳定，为恒定区(Fc区)。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(LCVR)和重链可变区(HCVR)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1、LCDR2、和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。本发明所述的抗体或抗原结合片段的LCVR区和HCVR区的CDR氨基酸残基在数量和位置符合已知的Kabat编号规则(LCDR1-3，HCDE2-3)，或者符合kabat和chothia的编号规则(HCDR1)。
本发明的抗体包括鼠源抗体、嵌合抗体、人源化抗体，优选人源化抗体。
术语“鼠源抗体”在本发明中为根据本领域知识和技能制备的对人PCSK9的 单克隆抗体。制备时用PCSK9抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。在本发明一个优选的实施方案中，所述的鼠源PCSK9抗体或其抗原结合片段，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，或进一步包含鼠源IgG1、IgG2、IgG3或其变体的重链恒定区。
术语“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要选建立分泌鼠源性特异性单抗的杂交瘤，然后从小鼠杂交瘤细胞中克隆可变区基因，再要据需要克隆人抗体的恒定区基因，将小鼠可变区基因与人恒定区基因连接成嵌合基因后插入人载体中，最后在真核工业系统或原核工业系统中表达嵌合抗体分子。在本发明一个优选的实施方案中，所述的PCSK9嵌合抗体的抗体轻链进一步包含人源κ、λ链或其变体的轻链Fc区。所述的PCSK9嵌合抗体的抗体重链进一步包含人源IgG1、IgG2、IgG3、IgG4或其变体的重链Fc区，优选包含人源IgG1、IgG2或IgG4重链恒定区，或者使用氨基酸突变(如YTE突变)后延长抗体在血清中的半衰期的IgG1、IgG2或IgG4变体。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将小鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体框架序列中产生的抗体。可以克服嵌合抗体由于携带大量小鼠蛋白成分，从而诱导的强烈的抗体可变抗体反应。此类框架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库(在因特网www.mrccpe.com.ac.uk/vbase可获得)，以及在Kabat，E.A.等人，1991Sequences of Proteins of Immunological Interest，第5版中找到。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。本发明的人源化抗体也包括进一步由噬菌体展示对CDR进行亲和力成熟后的人源化抗体。在本发明一个优选的实施方案中，所述的PCSK9人源化抗体小鼠的CDR序列选自SEQ ID NO:12,13,14,15,16或17；人的抗体可变区框架经过设计选择，其中所述抗体轻链可变区上的轻链FR区序列，来源于人种系轻链IGKV1-39*01和hjk2.1的组合序列；其中所述抗体重链可变区上的重链FR区序列，来源于人种系重链IGHV1-2*02和hjh2的组合序列。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区可进行最少反向突变，以保持活性。
本发明中所述的“抗原结合片段”，指具有抗原结合活性的Fab片段，Fab’片段，F(ab’)2片段，以及与人PCSK9结合的Fv片段ScFv片段；其包含选自SEQ ID NO:12至SEQ ID NO:17中的一个或多个本发明抗体的CDR区。Fv片段含有抗体重链可变区和轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般地，Fv抗体还包含在VH和VL结构域之间的多肽接头，且能够形 成抗原结合所需的结构。也可以用不同的连接物将两个抗体可变区连接成一条多肽链，称为单链抗体(single chain antibody)或单链Fv(sFv)。本发明的术语“与PCSK9结合”，指能与人PCSK9相互作用。本发明的术语“抗原结合位点”指抗原上不连续的，由本发明抗体或抗原结合片段识别的三维空间位点。
术语“Fc区”在本文中用于定义免疫球蛋白重链的C端区域，所述区域包含至少一部分的恒定区。该术语包括天然序列Fc区和变体Fc区。在某些实施方案中，人IgG重链Fc区从Cys226或Pro230延伸至重链的羰基端。然而，Fc区的C端赖氨酸(Lys447)可以存在或者可以不存在。除非另外说明，Fc区或恒定区中的氨基酸残基的编号是根据EU编号系统，其也被称为EU索引，如在Kabat等，Sequences of Proteins of Immunological Interest(免疫学感兴趣的蛋白质的序列)，5th Ed.Public Health Service，National Institutes of Health，Bethesda，MD，1991中所述。Fc区域是抗体的效应子功能所必需的。效应子功能包括启动补体依赖的细胞毒性(CDC)、启动吞噬作用和抗体依赖的细胞介导的细胞毒性(ADCC)并通过胞转作用转运抗体通过细胞屏障。此外，Fc区域对维持IgG类抗体的血清半衰期至关重要(Ward和Ghetie，Ther.Immunol.2:77-94(1995))。研究发现IgG抗体的血清半衰期由Fc和新生Fc受体(FcRn)的结合来介导。FcRn是由跨膜α链和可溶性β链(β2-微球蛋白)组成的异源二聚体。美国专利号6,165,745公开了一种通过将突变引入编码抗体的DNA片段生产生物半衰期减少的抗体的方法。该突变包括在Fc-绞链结构域的位置253、310、311、433或434处的氨基酸取代。美国专利号6,277,375B1公开了含有突变型IgG分子的组合物，该分子相对野生型IgG血清半衰期增加，其中该突变型IgG分子含有以下氨基酸取代：在252位苏氨酸取代亮氨酸，在254位苏氨酸取代丝氨酸，或在256位苏氨酸取代苯丙氨酸(M252Y、S254T和T256E)。也公开了在位置433、435或436处具有氨基酸取代的突变型IgG。美国专利号6,528,624公开了含有IgG Fc区域的一种抗体的变体，该变体在人IgG Fc区域的一个或多个氨基酸位置(位置270、322、326、327、329、331、333和334)具有氨基酸取代。PCT公开号WO 02/060919A2公开了修饰的IgG，该修饰的IgG包含的IgG恒定区相对于野生型IgG恒定区含有一个或多个氨基酸修饰，其中该修饰的IgG与含有野生型IgG恒定区的IgG相比增加了半衰期，并且其中一个或多个氨基酸修饰位于以下一个或多个位置：251、253、255、285-290、308-314、385-389、和428-435。具体地，本文所述“YTE”或“YET突变”指IgG1的Fc区的一个突变组合，用于促进Fc区与人FcRn的结合，延长抗体在人血清中的半衰期。YTE突变子包含三个“YTE突变子”的组合：M252Y、S254T和T256E，残基编号是根据EU编号系统，其也被称为EU索引，如在Kabat等(参考U.S.专利No.7,658,921所述)对IgG重链进行编号。相较于野生型抗体，YTE突变抗体大大延长了抗体在血清中的半衰期，如e.g.,Dall’Acqua et al,J.Biol.Chem.281:23514-24(2006)和U.S.专利号No.7,083,784。
现有技术中熟知生产和纯化抗体和抗原结合片段的方法，如冷泉港的抗体实验技术指南，5-8章和15章。例如，老鼠可以用人PCSK9或其片段免疫，所得到的抗体能被复性、纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样可以用常规方法制备。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人源FR区。人FR种系序列可以通过比对IMGT人类抗体可变区种系基因数据库和MOE软件，从ImMunoGeneTics(IMGT)的网站http://imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
本发明工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链(SEQ ID NO:28)和轻链(SEQ ID NO:30)的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端位点。通过表达与人PCSK9特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化。比如，用含调整过的缓冲液的A或G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再用PH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
“给予”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“给予”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予患者内用或外用治疗剂，例如包含本发明的任一种结合化合物的组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，以诱导这类症状退化或抑制这类症状发展到任何临床右测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本发明的实施方案(例如治疗方法或制品)在缓解每个目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和 Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的患者中应当减轻目标疾病症状。
“保守修饰”或“保守置换或取代”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecular Biology of the Gene,The Benjamin/Cummings Pub.Co.,第224页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。
“有效量”包含足以改善或预防医学疾病的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定患者或兽医学受试者的有效量可依据以下因素而变化：例如，待治疗的病症、患者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
“外源性”指根据情况在生物、细胞或人体外产生的物质。“内源性”指根据情况在细胞、生物或人体内产生的物质。
“同源性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源。一般而言，当比对两个序列而得到最大的同源性百分率时进行比较。
本文使用的表述“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括后代。因此，单词“转化体”和“转化细胞”包括原代受试细胞和由其衍生的培养物，而不考虑转移数目。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。在意指不同名称的情况下，其由上下文清楚可见。
本文使用的“聚合酶链式反应”或“PCR”是指其中微量的特定部分的核酸、RNA和/或DNA如在例如美国专利号4,683,195中所述扩增的程序或技术。一般来说，需要获得来自目标区域末端或之外的序列信息，使得可以设计寡核苷酸引物；这些引物在序列方面与待扩增模板的对应链相同或相似。2个引物的5’末端核苷酸可以与待扩增材料的末端一致。PCR可用于扩增特定的RNA序列、来自总基因组DNA的特定DNA序列和由总细胞RNA转录的cDNA、噬菌体或质粒序列等。一般参见Mullis等(1987)Cold Spring Harbor Symp.Ouant.Biol.51:263；Erlich编辑， (1989)PCR TECHNOLOGY(Stockton Press,N.Y.)。本文使用的PCR被视为用于扩增核酸测试样品的核酸聚合酶反应法的一个实例，但不是唯一的实例，所述方法包括使用作为引物的已知核酸和核酸聚合酶，以扩增或产生核酸的特定部分。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生的场合。例如，“任选包含1-3个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，所述其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
实施例与测试例
以下结合实施例进一步描述本发明，但这些实施例并非限制着本发明的范围。本发明实施例中未注明具体条件的实验方法，通常按照常规条件，如冷泉港的抗体技术实验手册，分子克隆手册；或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1、PCSK9抗原及检测用蛋白的制备
蛋白设计及表达
以UniProt Proprotein convertase subtilisin/kexin type 9(人PCSK9，Uniprot号：Q8MBP7)作为本发明PCSK9的模板，设计本发明涉及的抗原及检测用蛋白的氨基酸序列，可选的在PCSK9蛋白基础上融合不同的标签如his标签或促进免疫的肽段如PADRE肽，分别克隆到pTT5载体上(Biovector，Cat#:102762)或pTargeT载体上(promega,A1410)，在293细胞瞬转表达或CHO-S稳定表达，纯化，获得编码本发明抗原及检测用蛋白。
带His标签的PCSK9：PCSK9-His6，用于免疫原免疫小鼠或检测试剂
Figure PCTCN2016111053-appb-000001
Figure PCTCN2016111053-appb-000002
注释：划横线部分为信号肽，斜体部分为His6-tag(6组氨酸标签)。
带PADRE肽和His标签的PCSK9：PCSK9-PADRE-His6，作为免疫原，所含PADRE肽可以促进免疫；
Figure PCTCN2016111053-appb-000003
注释：划横线部分为信号肽，双划线部分为linker，点划线部分为PADRE肽，斜体部分为His6-tag。
带TEV酶切位点的PCSK9与his标签融合蛋白：PCSK9-TEV-His6，可通过TEV酶切获得N-PCSK9(N端pCSK9结构域)，作为免疫原；
Figure PCTCN2016111053-appb-000004
注释：划横线部分为信号肽，双划线部分为TEV酶切位点，斜体部分为His6-tag。
PCSK9-D374Y突变蛋白，带his标签：PCSK9-D374Y-His6，作为检测试剂；
Figure PCTCN2016111053-appb-000005
注释：划横线部分为信号肽，斜体部分为His6-tag。
PCSK9：插入生物素接受肽BP15及his标签的PCSK9蛋白：PCSK9-BP15-His6，作为检测试剂，BP15肽位置在表达过程中能够进行生物素标记，免除体外生物素标记及可能导致的构象变化；
Figure PCTCN2016111053-appb-000006
注释：划横线部分为信号肽，双划线部分为生物素接受肽，斜体部分为His6-tag。
PCSK9-Y：插入生物素接受肽及his标签的PCSK9D374Y突变体蛋白：PCSK9-D374Y-BP15-His6，检测蛋白；
Figure PCTCN2016111053-appb-000007
Figure PCTCN2016111053-appb-000008
注释：划横线部分为信号肽，双划线部分为生物素接受肽，斜体部分为His6-tag。
带Flag标签和His标签的pCSK9受体蛋白LDLR胞外域片段：LDLR-ECD-Flag-His6，检测试剂
Figure PCTCN2016111053-appb-000009
注释：划横线部分为信号肽，双划线部分为Flag标签，斜体部分为His6-tag；
LDLR-Fc：缩短形式的LDLR胞外域片段与hIgG1-Fc融合蛋白(具有与PCSK9结合活性)：LDLR-sECD–Fc(hIgG1)作为检测试剂
Figure PCTCN2016111053-appb-000010
注释：划横线部分为信号肽，双划线部分为缩短形式的具有与PCSK9结合活性的LDLR胞外域片段(LDLR-sECD)，斜体部分为hIgG1-Fc部分；
更加缩短形式的LDLR胞外域片段与hIgG1-Fc融合蛋白(具有与pCSK9结合活性)：LDLR-ssECD–Fc(hIgG1)作为检测试剂
Figure PCTCN2016111053-appb-000011
Figure PCTCN2016111053-appb-000012
注释：划横线部分为信号肽，双划线部分为更加缩短形式的具有与PCSK9结合活性的LDLR胞外域片段(LDLR-ssECD)，斜体部分为hIgG1-Fc部分。
实施例2、PCSK9、LDLR相关重组蛋白的纯化重组蛋白以及杂交瘤抗体、重组抗体的纯化
1、带His标签的重组蛋白的纯化步骤：
将细胞表达上清样品高速离心去除杂质，并将缓冲液换置换为PBS，加入咪唑至终浓度为5mM。用含有5mM咪唑的PBS溶液平衡镍柱，冲洗2-5倍柱体积。将置换后的上清样品上IMAC柱。用含有5mM咪唑的PBS溶液冲洗柱子，至A280读数降至基线。后用PBS+10mM咪唑冲洗层析柱，除去非特异结合的杂蛋白，并收集流出液。再用含有300mM咪唑的PBS溶液洗脱目的蛋白，并收集洗脱峰。收集的洗脱液浓缩后用凝胶层析Superdex200(GE)进一步纯化，流动相为PBS。去聚体峰，收集洗脱峰。所得到的蛋白经电泳，肽图，LC-MS鉴定为正确后分装备用。得到带His标签的PCSK9-His6(SEQ ID NO：1)、PCSK9-PADRE-His6(SEQ ID NO：2)、PCSK9-TEV-His6(SEQ ID NO：3)PCSK9-D374Y-His6(SEQ ID NO：4)、PCSK9-BP15-His6(SEQ ID NO：5)、PCSK9-D374Y-BP15-His6(SEQ ID NO：6)用于本发明抗体的免疫原或检测试剂。其中PCSK9-TEV-His6纯化后通过TEV酶进行酶切，酶切产物再利用IMAC柱结合去除TEV酶、未酶切完全的PCSK9-TEV-His6或切除的带His标签的C端结构域片段，IMAC流出液中浓缩获得仅留N端结构域的PCSK9片段(缩写为N-PCSK9)，作为免疫原用于小鼠免疫。
2、带His标签和Flag标签的LDLR-ECD-Flag-His6(SEQ ID NO:7)重组蛋白的纯化步骤：
将样品高速离心去除杂质，并浓缩至适当体积。利用0.5×PBS平衡flag亲和柱，冲洗2-5倍柱体积。将除杂后的细胞表达上清样品上柱。用0.5×PBS冲洗柱子，至A280读数降至基线。用含有0.3M NaCl的PBS冲洗柱子，冲洗杂蛋白，并收集。用0.1M乙酸(pH3.5-4.0)洗脱目的蛋白，并收集，调节pH至中性。收集的洗脱液浓缩后用凝胶层析Superdex200(GE)进一步纯化，流动相为PBS。去聚体峰，收集洗脱峰收集样品经电泳，肽图，LC-MS鉴定正确后分装备用。得到带FLAG/His6标签的LDLR-ECD-Flag-His6(SEQ ID NO:7),用于本发明抗体的性能测试。
3、LDLR的Fc融合蛋白的纯化步骤：
将细胞表达上清样品高速离心去除杂质，浓缩至适当体积后上Protein A柱。用PBS冲洗柱子，至A280读数降至基线。用100mM sodium acetate pH3.0洗脱目的蛋白，用1M Tris-HCl中和。洗脱样品适当浓缩后利用PBS平衡好的凝胶层析Superdex200(GE)进一步纯化，去聚体的峰收集好后分装备用。此方法用来纯化LDLR-sECD–Fc(hIgG1)(SEQ ID NO:8)和LDLR-ssECD–Fc(hIgG1)(SEQ ID NO:9)。两者可用作PCSK9抗体功能性测试。
实施例3、抗人PCSK9杂交瘤单克隆抗体的制备
1、免疫
抗人PCSK9单克隆抗体通过免疫小鼠产生。实验用SJL白小鼠，雌性，6周龄(北京维通利华实验动物技术有限公司，动物生产许可证号：SCXK(京)2012-0001)。饲养环境：SPF级。小鼠购进后，实验室环境饲养1周，12/12小时光/暗周期调节，温度20-25℃；湿度40-60％。将已适应环境的小鼠按两种方案免疫(A/B)，每组6-10只。免疫抗原为带His标签的人PCSK9-His6(SEQ ID NO:1)、PCSK9-PADRE-His6(SEQ ID NO:2)及N-PCSK9(SEQ ID NO:3)。
方案A用弗氏佐剂(sigma Lot Num：F5881/F5506)乳化：首免用弗氏完全佐剂(CFA)，其余加强免疫用弗氏不完全佐剂(IFA)。抗原与佐剂比例为1:1，100μg/只(首免)，50μg/只(加强免疫)。第0天腹膜内(IP)注射100μg/只的乳化后抗原，首免后每两周一次，共6-8周。
方案B用Titermax(sigma Lot Num：T2684)与Alum(Thremo Lot Num：77161)交叉免疫。抗原与佐剂(titermax)比例为1:1，抗原与佐剂(Alum)比例为3:1，10-20μg/只(首免)，5μg/只(加强免疫)。第0天腹膜内(IP)注射20/10μg/只的乳化后抗原，首免后每周一次，Titermax和Alum交替使用，共6-11周。免疫四周后，根据背部结块和腹部肿胀情况，选择背部或腹膜内注射抗原。
2、细胞融合
选择血清中抗体滴度高(见后面的测试例1和2，结合PCSK9的ELISA方法)并且滴度趋于平台的小鼠进行脾细胞融合，融合前72小时冲刺免疫所选小鼠，PCSK9-His6 10μg/只，腹腔注射。采用优化的PEG介导的融合步骤将脾淋巴细胞与骨髓瘤细胞Sp2/0细胞(
Figure PCTCN2016111053-appb-000013
CRL-8287TM)进行融合得到杂交瘤细胞。融合好的杂交瘤细胞用HAT完全培养基(含20％FBS、1×HAT和1×OPI的RPMI-1640培养基)重悬，分装于96孔细胞培养板中(1×105/150μl/孔)，37℃，5％CO2孵育。融合后的第5天加入HAT完全培养基，50μl/孔，37℃，5％CO2孵育。融合后第7天～8天，根据细胞生长密度，全换液，培养基为HT完全培养基(含20％FBS、1×HT和1×OPI的RPMI-1640培养基)，200μl/孔，37℃，5％CO2孵育。
3、杂交瘤细胞筛选
融合后第10-11天，根据细胞生长密度，进行结合PCSK9或PCSK9-Y的ELISA方法检测(见测试例1和2)。并将结合ELISA检测的阳性孔细胞进行PCSK9或PCSK9-Y与LDLR结合的阻断ELISA检测(见测试例3和4)，阳性孔换液，并根据细胞密度及时扩大至24孔板中。移入24孔板的细胞株经过复测后进行保种和第一次亚克隆。第一次亚克隆筛选(见测试例1和2)为阳性的进行保种，并进行第二次亚克隆。第二次亚克隆为阳性(见测试例1和2)的进行保种和蛋白表达。多次融合获得有阻断PCSK9或PCSK9-Y与LDLR结合效果(见测试例3和4)的杂交瘤细胞。
通过阻断实验和结合实验筛选得到杂交瘤克隆mAb-001，用无血清细胞培养法进一步制备抗体，按纯化实例纯化抗体，供在检测例中使用。
其中测得杂交瘤克隆mAb-001的鼠抗可变区序列如下：
>mAb-001VH
Figure PCTCN2016111053-appb-000014
>mAb-001VL
Figure PCTCN2016111053-appb-000015
注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列，下划线为CDR序列。
表1各重链及轻链CDR区序列
Figure PCTCN2016111053-appb-000016
实施例4、抗人PCSK9杂交瘤单克隆抗体的人源化
1、杂交瘤克隆mAb-001人源化框架选择
通过比对IMGT人类抗体重轻链可变区种系基因数据库和MOE软件，分别挑选与mAb-001同源性高的重轻链可变区种系基因作为模板，将这两个鼠源抗体的CDR分别移植到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。其中氨基酸残基由Kabat编号系统确定并注释。
鼠源抗体mAb-001的人源化轻链模板为IGKV1-39*01和hjk2.1，人源化重链模板为IGHV1-2*02和hjh2，人源化后得到人源化抗体h001-1的可变区序列如下：
>h001-1VH
Figure PCTCN2016111053-appb-000017
>h001-1VL
Figure PCTCN2016111053-appb-000018
注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列，下划线为CDR序列。
2、杂交瘤克隆mAb-001的模板选择和回复突变设计，见下表2；杂交瘤克隆回复突变后的人源化序列组合见表4。
表2.杂交瘤克隆回复突变设计
Figure PCTCN2016111053-appb-000019
注：如S66D表示依照Kabat编号系统，将66位S突变回D。
Grafted代表鼠抗体CDR植入人种系FR区序列，各突变可变区具体序列如下表3：
表3
Figure PCTCN2016111053-appb-000020
注：序列中横线部分为CDR区。
表4：鼠抗mAb-001人源化序列组合
  h001_VL.1 h001_VL.1A h001_VL.1B h001_VL.1C
h001_VH.1 h001-1 h001-2 h001-3 h001-4
h001_VH.1A h001-5 h001-6 h001-7 h001-8
h001_VH.1B h001-9 h001-10 h001-11 h001-12
h001_VH.1C h001-13 h001-14 h001-15 h001-16
h001_VH.1D h001-17 h001-18 h001-19 h001-20
h001_VH.1E h001-21 h001-22 h001-23 h001-24
注：该表表示各种序列及其突变序列组合所得的人源化抗体可变区部分的组合。如h001-1表示，人源化抗体h001-1的可变区由轻链h001_VL1、重链h001_VH.1A组成。其它类推。
3、将以上的人源化序列组合进行抗体化，重链恒定区来自人IgG1，轻链恒定区来自人kappa链。得到相应的人源化抗体，进行结合pCSK9的ELISA方法检测(见测试例1)，和结合pCSK9-Y的ELISA方法检测(见测试例2)；并将结合ELISA检测的阳性孔细胞进行pCSK9/LDLR结合的阻断ELISA检测(见测试例4)，和进行pCSK9-Y/LDLR结合的阻断ELISA检测(见测试例3)；结果见表5-8。
结果显示，本发明得到的PCSK9抗体与PCSK9和PCSK9-Y有较高的结合活性；并且能有效阻断PCSK9/PCSK9-Y与LDLR之间的结合。
实施例5、构建和表达抗人PCSK9人源化抗体IgG1及IgG1-YTE形式
本发明构建和表达抗人PCSK9人源化抗体的方法如下：
1、引物设计：利用在线软件DNAWorks(v3.2.2)(http://helixweb.nih.gov/dnaworks/)设计多条引物合成VH/VK含重组所需基因片段：5’-30bp Signal peptide+VH/VK+30bp CH1/CL-3’。引物设计原则：目的基因2与目的基因1有2个aa不一样，则另设突变位点所在引物，如图1所示。
2、片段拼接：按照TaKaRa公司Primer STAR GXL DNA聚合酶操作说明书，用上面设计的多条引物，分两步PCR扩增得到VH/VK含重组所需基因片段。
3、表达载体pHr(带信号肽及恒定区基因(CH1-FC/CL)片段)构建及酶切
利用一些特殊的限制性内切酶,如BsmBI，识别序列与酶切位点不同的特性设计构建表达载体pHr(带信号肽及恒定区基因(CH1-FC/CL)片段)，如图2所示。BsmBI酶切载体，切胶回收备用。
4、重组构建表达载体VH-CH1-FC-pHr/VK-CL-pHr
VH/VK含重组所需基因片段与BsmBI酶切回收表达载体pHr(带信号肽及恒定区基因(CH1-FC/CL)片段)按3:1比例分别加入DH5α感受态细胞中，0℃冰浴30min，42℃热击90秒，加入5倍体积LB medium，37℃孵育45min，涂布LB-Amp平板，37℃培养过夜，挑取单克隆送测序得到各目的克隆。
本发明的抗体可以，但不限于以上设计构建方式。以h001-4为例进行抗体及其突变体设计，得到①h001-4-WT：h001-4的IgG1形式，即人源化序列组合h001-4，结合来自人IgG1的重链恒定区，与来自人kappa链的轻链恒定区；②h001-4-YTE：h001-4-IgG1-YTE形式，即人源化序列组合h001-4，结合突变的人IgG1(YTE突变)的重链恒定区，与来自人kappa链的轻链恒定区。突变的人IgG1也可以是别种形式的突变。得到的抗体及突变的抗体用BIAcore检测其亲和力(测试例6)，结果见表9。
本发明构建和表达抗人PCSK9人源化抗体(IgG1及IgG1-YTE形式)序列如下：
h001-4IgG1形式，重链恒定区来自人IgG1，轻链恒定区来自人kappa轻链：
重链氨基酸序列(人IgG1)：
Figure PCTCN2016111053-appb-000021
重链DNA序列：
Figure PCTCN2016111053-appb-000022
Figure PCTCN2016111053-appb-000023
h001-4-kappa
轻链氨基酸序列：
Figure PCTCN2016111053-appb-000024
轻链DNA序列：
Figure PCTCN2016111053-appb-000025
h001-4-IgG1-YTE(轻链为h001-4-kappa:SEQ ID NO：30)
重链氨基酸序列：IgG1-YTE
Figure PCTCN2016111053-appb-000026
重链DNA序列：
Figure PCTCN2016111053-appb-000027
备注：下划线部分为信号肽DNA序列
以下用生化测试方法验证本发明性能及有益效果。
测试例1、PCSK9抗体结合野生型PCSK9蛋白的ELISA实验
本发明PCSK9抗体与PCSK9的结合力测试，通过抗体与固定在ELISA板上野生型PCSK9(WT-PCSK9，SEQ ID NO:5)的结合的量来检测。
用PBS稀释链霉亲和素(sigma，CAT#S4762)至2μg/ml，包被在96孔ELISA板上，4℃放置过夜。洗板后，在37℃用Tris缓冲液(含0.9mM氯化钙、0.05％Tween 20和5％脱脂奶粉)封闭2小时。洗板，加入内部生产的生物素标记的PCSK9(bio-WT-PCSK9，用含0.9mM氯化钙、0.05％Tween 20和1％脱脂奶粉的Tris缓 冲液稀释)100μl/孔，37℃孵育1小时。洗板，加入不同浓度稀释的抗PCSK9抗体样品，37℃孵育1小时。再洗板，加入辣根过氧化物酶-羊抗人(H+L)抗体(jackson，CAT#109-035-088)，37℃孵育1小时。再洗板，加入四甲基联苯胺溶液显色。最后加入终止液，在酶标仪上测量OD450，并计算其EC50值。
本发明嵌合抗体、回复突变后抗体与PCSK9的结合力ELISA实验，结果见表5。
表5.本发明PCSK9抗体与PCSK9的结合活性测试
克隆号 EC50(μg/ml)
h001-1 0.0084
h001-2 0.0123
h001-3 0.0113
h001-4 0.012
h001-5 0.0141
h001-6 0.01
h001-7 0.012
h001-8 0.009
h001-9 0.0136
h001-10 0.0176
h001-11 0.0129
h001-12 0.0103
h001-13 0.0071
h001-14 0.0084
h001-15 0.011
h001-16 0.0082
h001-17 0.0114
h001-18 0.0147
h001-19 0.0139
h001-20 0.0126
h001-21 0.0145
h001-22 0.0123
h001-23 0.0118
h001-24 0.0092
Ch-001 0.0084
结果显示，本发明PCSK9抗体与PCSK9有较高的结合活性。
测试例2、PCSK9抗体结合PCSK9-Y的ELISA实验
本发明PCSK9抗体与PCSK9-Y的结合力测试，通过抗体与固定在ELISA板上PCSK9-Y(突变型PCSK9，SEQ ID NO:6)的结合的量来检测。
用PBS稀释链霉亲和素(sigma，CAT#S4762)至2μg/ml，包被在96孔ELISA板上，4℃放置过夜。洗板后，在37℃用Tris缓冲液(含0.9mM氯化钙、0.05％Tween 20和5％脱脂奶粉)封闭2小时。洗板，加入内部生产的生物素标记的PCSK9-Y(bio-PCSK9-Y，用含0.9mM氯化钙、0.05％Tween 20和1％脱脂奶粉的Tris缓冲液稀释)100μl/孔，37℃孵育1小时。洗板，加入不同浓度稀释的抗PCSK9抗体样品，37℃孵育1小时。再洗板，加入辣根过氧化物酶-羊抗人(H+L)抗体(jackson，CAT#109-035-088)，37℃孵育1小时。再洗板，加入四甲基联苯胺溶液显色。最后加入终止液，在酶标仪上测量OD450，并计算其EC50值。
本发明嵌合抗体、回复突变后抗体与突变型PCSK9的结合力ELISA实验，结果见表6。
表6.本发明PCSK9抗体与PCSK9-Y的结合活性测试
克隆号 EC50(μg/ml)
h001-1 0.0132
h001-2 0.0157
h001-3 0.0152
h001-4 0.0179
h001-5 0.0152
h001-6 0.0144
h001-7 0.0137
h001-8 0.0165
h001-9 0.0194
h001-10 0.0209
h001-11 0.0170
h001-12 0.0124
h001-13 0.0096
h001-14 0.0112
h001-15 0.0178
h001-16 0.0111
h001-17 0.0161
h001-18 0.0191
h001-19 0.0204
h001-20 0.0170
h001-21 0.0119
h001-22 0.0111
h001-23 0.0125
h001-24 0.0170
Ch-001 0.0132
结果显示，本发明PCSK9抗体与PCSK9-Y有较高的结合活性。
测试例3、PCSK9抗体对LDLR-FC/PCSK9-Y结合的阻断
抗PCSK9抗体对LDLR-FC(SEQ ID NO:8)和PCSK9-Y(突变型PCSK9，SEQ ID NO:6)结合的阻断能力测试，通过检测在抗体存在的条件下，PCSK9-Y与LDLR结合的量来确定。
用磷酸缓冲液稀释LDLR-FC，至2μg/ml，包被在96孔ELISA板(Costar，CAT#3590)上，4℃放置过夜。洗板后，在37℃用Tris缓冲液(含0.9mM氯化钙、0.05％Tween 20和5％脱脂奶粉)封闭2小时。洗板，加入生物素标记的PCSK9-Y(bio-PCSK9-Y，用含0.9mM氯化钙、0.05％Tween 20和1％脱脂奶粉的Tris缓冲液稀释至终浓度1μg/ml)，和抗体样品(用含0.9mM氯化钙、0.05％Tween 20和1％脱脂奶粉的Tris缓冲液稀释)的混合液100μl/孔，37℃孵育1小时。洗板，加入辣根过氧化物酶-链霉亲和素(sigma，CAT#S2438)，37℃孵育1小时。再洗板，加入四甲基联苯胺溶液显色。最后加入终止液，在酶标仪上测量OD450，并计算其IC50值。
本发明嵌合抗体、回复突变后抗体对LDLR-FC/PCSK9-Y结合的阻断效果测试，结果见表7：
表7.本发明PCSK9抗体阻断PCSK9-Y与LDLR之间结合的效果测试
克隆号 IC50(μg/ml)
h001-1 0.5658
h001-2 0.4553
h001-3 0.4749
h001-4 0.5302
h001-5 0.4677
h001-6 0.4374
h001-7 0.5150
h001-8 0.4145
h001-9 0.5203
h001-10 0.5142
Ch-001 0.3915
结果显示，本发明PCSK9抗体能有效阻断PCSK9-Y与LDLR之间的结合。
用上面所述方法，测试本发明PCSK9抗体对其它形式的LDLR-FC(内部生产的，序列见SEQ ID NO:7或SEQ ID NO:9)和PCSK9-Y(SEQ ID NO:5)结合的阻断能力，实验证明本发明PCSK9抗体能有效阻断PCSK9与缩短形式的LDLR之间的结合。
测试例4、PCSK9抗体对LDLR-FC/PCSK9结合的阻断
本发明PCSK9抗体对LDLR-FC(内部生产的，序列为SEQ ID NO:8)和PCSK9 (SEQ ID NO:5)结合的阻断能力测试，通过检测在抗体存在的条件下，PCSK9与LDLR结合的量来确定。
用磷酸缓冲液稀释LDLR-FC至5μg/ml，包被在96孔ELISA板上，4℃放置过夜。洗板后，在37℃用Tris缓冲液(含0.9mM氯化钙、0.05％Tween 20和5％脱脂奶粉)封闭2小时。洗板，加入生物素标记的PCSK9(bio-WT-PCSK9，用含0.9mM氯化钙、0.05％Tween 20和1％脱脂奶粉的Tris缓冲液稀释至终浓度2μg/ml)和抗体样品(用含0.9mM氯化钙、0.05％Tween 20和1％脱脂奶粉的Tris缓冲液稀释)的混合液100μl/孔，37℃孵育1小时。洗板，加入辣根过氧化物酶-链霉亲和素(sigma，CAT#S2438)，37℃孵育1小时。再洗板，加入四甲基联苯胺溶液显色。最后加入终止液，在酶标仪上测量OD450，并计算其IC50值。
本发明嵌合抗体、回复突变后抗体对LDLR-FC/PCSK9结合的阻断效果测试，结果见表8。
表8.本发明PCSK9抗体阻断PCSK9与LDLR之间结合的效果测试
克隆号 IC50(μg/ml)
h001-1 0.4997
h001-2 0.6750
h001-3 0.7021
h001-4 0.7597
h001-5 4.322
h001-6 0.6620
h001-7 0.6521
h001-8 0.7738
h001-9 0.9230
h001-10 0.8290
Ch-001 0.8363
结果显示，本发明PCSK9抗体能有效阻断PCSK9与LDLR之间的结合。
用上面所述方法，测试本发明PCSK9抗体对其它形式的LDLR-FC(内部生产的，序列见SEQ ID NO:7或SEQ ID NO:9)和PCSK9(SEQ ID NO:5)结合的阻断能力，实验证明本发明PCSK9抗体能有效阻断PCSK9与缩短形式的LDLR之间的结合。
测试例5、PCSK9抗体对LDL的摄取实验
HepG2细胞(中科院细胞库，#CAT，TCHu72)培养在DMEM培养基(Hyclone，#CAT SH30243.01B)中(含10％胎牛血清，Gibco，#CAT 10099-141)。当细胞覆盖80-90％时，消化吹散后计数1.5*104cells/孔铺于96孔板。24小时后，更换培养基为DMEM，10％无脂蛋白血清(Millipore，CAT#LP4)。48小时后，用磷酸缓冲液洗2次，加入在4℃预孵育1小时的含PCSK9(SEQ ID NO:1，终浓度10μg/ml) 和抗体样品(用培养基稀释至不同浓度)的混合物，以及终浓度10μg/ml的
Figure PCTCN2016111053-appb-000028
Figure PCTCN2016111053-appb-000029
(Invitrogen，CAT#L3483)，37℃孵育。6小时后，用磷酸缓冲液洗板2次，用酶标仪读取荧光值(EX485nm/EM535nm)。然后加入50μl/孔
Figure PCTCN2016111053-appb-000030
细胞活性发光检测试剂(Promega,G7571)，读取化学发光值。LDL uptake结果如图3，图4所示，数据结果显示本发明PCSK9抗体能够促进HepG2细胞摄取LDL。
测试例6、BIAcore检测PCSK9抗体亲和力实验
按照人Fab捕获试剂盒(Cat.#28-9583-25，GE)说明书中所述的方法，将人Fab捕获分子共价偶联于CM5生物传感芯片(Cat.#BR-1000-12，GE)上，从而亲和捕获待测抗体，然后于芯片表面流经人PCSK9抗原(带His标签的人PCSK9：PCSK9-His6，SEQ ID NO：1)，利用Biacore仪器实时检测反应信号从而获得结合和解离曲线，通过拟合得到亲和力数值，见表9。在实验中每个循环解离完成后，用人Fab捕获试剂盒(GE)里配置的再生溶液将生物芯片洗净再生。
表9：抗PCSK9抗体的亲和力
Figure PCTCN2016111053-appb-000031
本发明PCSK9抗体与人PCSK9抗原有强亲和力。
用上面相似的方法，检测本发明PCSK9抗体与PCSK9-Y(SEQ ID NO：4)的亲和力，显示本发明PCSK9抗体与PCSK9-Y抗原有较强亲和力。
测试例7、PCSK9抗体体内药效实验
本实验构建过表达人PCSK9的小鼠模型,进行尾静脉注射PCSK9抗体，来评价本发明PCSK9抗体在过表达人PCSK9的小鼠体内降低LDL-c的作用。人IgG(从混合的正常人血清中，利用传统的亲和层析方法如ProteinA纯化获得的人免疫球蛋白)作为空白对照。
C57Bl/6小鼠(购自上海西普尔·必凯实验动物有限责任公司)实验室环境适应5天，通过尾静脉注射AAV-PCSK9病毒(北京本元正阳基因技术有限公司)，注射4×1011v.g.。注射病毒后于实验前一天禁食过夜，眼眶取血，用HDL and LDL/VLDL Cholesterol Quantification Kit(购自BioVision公司，货号#K613-100)检测LDL-c，根据LDL-c浓度随机分组，每组6只小鼠(n＝6)，进行尾静脉注射给药，内部生产的人IgG、h001-4-WT抗体给药剂量为10mg/kg(人IgG、h001-4-WT抗体用PBS配制，浓度为1mg/ml)。取血前禁食6小时，给药后第24、48、72、96小时眼眶取血，37℃放置1小时，3500rpm离心10分钟，取血清保存在-80℃。
最后一次取血清后，把冻存的血清在同一天检测。用HDL and LDL/VLDL Cholesterol Quantification Kit检测血清中LDL-c浓度，按照试剂盒说明书操作。
实验结果如图5所示，正常小鼠血清LDL-c浓度约为12mg/dl。注射AAV8-PCSK9病毒后，血清中LDL-c浓度达平均40mg/dl。分组后给药，给药24小时后，与人IgG组相比，h001-4-WT组LDL-c浓度下降50％；给药48小时后，h001-4-WT组LDL-c浓度下降49％；给药72小时后，h001-4-WT组LDL-c浓度下降32％；给药96小时后，h001-4-WT组LDL-c浓度下降20％，如表10和图6所示。
综上，h001-4-WT能够降低过表达人PCSK9的小鼠血清中LDL-c浓度，且药效持续到72小时。
表10.各组小鼠血清中LDL-c浓度变化
Figure PCTCN2016111053-appb-000032
测试例8、竞争性实验
在竞争性ELISA实验中，我们将一种抗体包板过夜，之后同时加入生物素化的pCSK9-his和50倍于包板浓度的竞争抗体，包板抗体和溶液中的抗体将竞争性结合抗原，之后检测板上抗原的信号。结果显示，h001-4和21B12(US8030457B2)自身能够竞争性结合抗原外，h001-4和21B12之间无明显竞争结合，提示两者抗原表位的不同。
IR(％) h001-4 21B12
h001-4 95.97 0.42％
21B12 3.86 97.78
测试例9、食蟹猴体内药效及药代检测
为考察本发明的抗体在体内的作用效果及代谢情况，尝试了在食蟹猴体内给药实验，分别给药h001-4-WT及h001-4-YTE。采用静脉注射给药，剂量选择3mg/kg，每组3只雄性食蟹猴。约2～4mL/分钟，缓慢推注。通过不同时间点取血检测脂蛋白尤其是低密度脂蛋白(LDL)及血清中抗体浓度，其中脂蛋白检测点为给药前和给药后1、4、8、12、16、20、24、28天，PK采血点为给药前、给药后15分钟、30分钟、1小时、3小时、8小时、12小时、24小时、48小时、72、96、120小时、144小时、168小时、336小时、504小时、672小时。
试验结果显示(图7)h001-4-WT和h001-4-YTE均能够明显降低食蟹猴体内LDL的含量，且h001-4-YTE的降低持续时间要优于h001-4-WT。
药代取血点血清样品通过ELISA检测其中h001-4-WT和h001-4-YTE的含量，方法参考测试例1所述，结果显示h001-4-WT在食蟹猴体内半衰期为4天，而h001-4-YTE在食蟹猴体内半衰期为7.3天，YTE相比WT具有明显延长的体内半衰期。

Claims (27)

  1. 一种PCSK9抗体或其抗原结合片段，其包含一个或多个选自以下的CDR：如序列SEQ ID NO:12、SEQ ID NO:13或SEQ ID NO:14所示的、或与SEQ ID NO:12、SEQ ID NO:13或SEQ ID NO:14具有至少95％序列同一性的序列所示的HCDR；和如序列SEQ ID NO:15、SEQ ID NO:16或SEQ ID NO:17所示的、或与SEQ ID NO:15、SEQ ID NO:16或SEQ ID NO:17具有至少95％序列同一性的序列所示的LCDR。
  2. 如权利要求1所述的PCSK9抗体或其抗原结合片段，其包含分别如SEQ ID NO:12、SEQ ID NO:13和SEQ ID NO:14所示的HCDR1、HCDR2和HCDR3，或包含分别与SEQ ID NO:12、SEQ ID NO:13和SEQ ID NO:14具有至少95％序列同一性的序列所示的HCDR1、HCDR2和HCDR3。
  3. 如权利要求1所述的PCSK9抗体或其抗原结合片段，其包含分别如SEQ ID NO:15、SEQ ID NO:16和SEQ ID NO:17所示的LCDR1、LCDR2和LCDR3，或包含分别与SEQ ID NO:15、SEQ ID NO:16和SEQ ID NO:17具有至少95％序列同一性的序列所示的LCDR1、LCDR2和LCDR3。
  4. 如权利要求1-3任一项所述的PCSK9抗体或其抗原结合片段，其包含分别如SEQ ID NO:12、SEQ ID NO:13和SEQ ID NO:14所示的HCDR1、HCDR2和HCDR3，或包含分别与SEQ ID NO:12、SEQ ID NO:13和SEQ ID NO:14具有至少95％序列同一性的序列所示的HCDR1、HCDR2和HCDR3；
    和分别如SEQ ID NO:15、SEQ ID NO:16和SEQ ID NO:17所示的LCDR1、LCDR2和LCDR3，或包含分别与SEQ ID NO:15、SEQ ID NO:16和SEQ ID NO:17具有至少95％序列同一性的序列所示的LCDR1、LCDR2和LCDR3。
  5. 如权利要求1至4任一项所述的PCSK9抗体或其抗原结合片段，其中所述的PCSK9抗体或其抗原结合片段为鼠源抗体或其片段。
  6. 如权利要求1至4任一项所述的PCSK9抗体或其抗原结合片段，其中所述PCSK9抗体的轻链可变区进一步包含鼠源κ链或鼠源κ链变体的轻链FR区、或者鼠源λ链或鼠源λ链变体的轻链FR区；其中所述PCSK9抗体的重链可变区进一步包含鼠源IgG1或其变体的重链FR区、或IgG2或其变体的重链FR区、或IgG3或其变体的重链FR区。
  7. 如权利要求6所述的PCSK9抗体或其抗原结合片段，其包含SEQ ID NO: 10所示的重链可变区和SEQ ID NO:11所示的轻链可变区。
  8. 根据权利要求1至4任一项所述的PCSK9抗体或其抗原结合片段，其中所述PCSK9抗体的轻链进一步包含鼠源κ链或其变体的轻链恒定区、或者鼠源λ链或其变体的轻链恒定区；其中所述PCSK9抗体重链进一步包含鼠源IgG1或其变体的重链恒定区、或IgG2或其变体的重链恒定区、或IgG3或其变体的重链恒定区。
  9. 如权利要求1至4任一项所述的PCSK9抗体或其抗原结合片段，其中所述的PCSK9抗体或其抗原结合片段为嵌合抗体或其片段。
  10. 如权利要求1至4任一项所述的PCSK9抗体或其抗原结合片段，其中所述的抗体或其抗原结合片段为人源化抗体或其片段。
  11. 如权利要求10所述的PCSK9抗体或其抗原结合片段，其中所述的人源化抗体重链可变区上的重链FR区序列来源于人种系重链，IGHV1-2*02和hjh2的组合序列或其突变序列；所述人源化抗体包含人种系重链IGHV1-2*02的FR1、FR2、FR3区和hjh2的FR4区或其突变序列。
  12. 如权利要求11所述的PCSK9抗体或其抗原结合片段，其中所述的人源化抗体含有SEQ ID NO:18所示的重链可变区；或SEQ ID NO:18变体所示的重链可变区；其中所述SEQ ID NO:18变体是在SEQ ID NO:18所示的重链可变区位置上具有1-10个氨基酸变化的序列。
  13. 如权利要求12所述的PCSK9抗体或其抗原结合片段，其中所述的SEQ ID NO:18变体是在SEQ ID NO:18所示的重链可变区的FR区位置上具有1-10个氨基酸的回复突变；优选的，所述回复突变选自T30N,R87T,R72A,T74K,M48I,V68A,M70L,R38K和R67K的氨基酸回复突变。
  14. 如权利要求11所述的PCSK9抗体或其抗原结合片段，其中所述人源化抗体包含选自SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22和SEQ ID NO:23的序列所示的重链可变区。
  15. 如权利要求10所述的PCSK9抗体或其抗原结合片段，其中所述的人源化抗体轻链可变区上的轻链FR区序列来源于人种系轻链模板IGKV1-39*01和hjk2.1的组合序列或其突变序列；所述的人源化抗体包含人种系轻链IGKV1-39*01的FR1、FR2、FR3区和hjk2.1的FR4区或其突变序列。
  16. 如权利要求15所述的PCSK9抗体或其抗原结合片段，其中所述人源化抗体进一步包含SEQ ID NO:24所示的、或SEQ ID NO:24变体所示的轻链可变区；所述的SEQ ID NO:24变体是在SEQ ID NO:24所示的轻链可变区位置上具有1-10的氨基酸变化的序列。
  17. 如权利要求16所述的PCSK9抗体或其抗原结合片段，其中所述的SEQ ID NO:24变体是在SEQ ID NO:24所示的轻链可变区的FR区位置上具有1-10个氨基酸的回复突变；优选的，所述回复突变选自T5S,S66D,Q3V和A49S的氨基酸回复突变；优选为A43S的氨基酸变化。
  18. 如权利要求15所述的PCSK9抗体或其抗原结合片段，其中所述人源化抗体包含选自SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27的序列所示的轻链可变区。
  19. 如权利要求10所述的PCSK9抗体或其抗原结合片段，其中所述人源化抗体包含重链可变区序列和/或轻链可变区序列，所述重链可变区序列选自SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22和SEQ ID NO:23所示的序列，或所述重链可变区序列选自与SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22和SEQ ID NO:23具有至少95％序列同一性的序列；所述轻链可变区序列选自SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27所示的序列，或所述轻链可变区序列选自选自与SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27具有至少95％序列同一性序列。
  20. 如权利要求10所述的PCSK9抗体或其抗原结合片段，其中所述的PCSK9抗体包含选自以下的重链可变区和轻链可变区：
    1)SEQ ID NO:18的重链可变区和SEQ ID NO:25的轻链可变区，
    2)SEQ ID NO:18的重链可变区和SEQ ID NO:26的轻链可变区，
    3)SEQ ID NO:18的重链可变区和SEQ ID NO:27的轻链可变区，
    4)SEQ ID NO:19的重链可变区和SEQ ID NO:24的轻链可变区，
    5)SEQ ID NO:19的重链可变区和SEQ ID NO:25的轻链可变区，
    6)SEQ ID NO:19的重链可变区和SEQ ID NO:26的轻链可变区，
    7)SEQ ID NO:19的重链可变区和SEQ ID NO:27的轻链可变区，
    8)SEQ ID NO:20的重链可变区和SEQ ID NO:24的轻链可变区，
    9)SEQ ID NO:20的重链可变区和SEQ ID NO:25的轻链可变区，
    10)SEQ ID NO:20的重链可变区和SEQ ID NO:26的轻链可变区，
    11)SEQ ID NO:20的重链可变区和SEQ ID NO:27的轻链可变区，
    12)SEQ ID NO:21的重链可变区和SEQ ID NO:24的轻链可变区，
    13)SEQ ID NO:21的重链可变区和SEQ ID NO:25的轻链可变区，
    14)SEQ ID NO:21的重链可变区和SEQ ID NO:26的轻链可变区，
    15)SEQ ID NO:21的重链可变区和SEQ ID NO:27的轻链可变区，
    16)SEQ ID NO:22的重链可变区和SEQ ID NO:24的轻链可变区，
    17)SEQ ID NO:22的重链可变区和SEQ ID NO:25的轻链可变区，
    18)SEQ ID NO:22的重链可变区和SEQ ID NO:26的轻链可变区，
    19)SEQ ID NO:22的重链可变区和SEQ ID NO:27的轻链可变区，
    20)SEQ ID NO:23的重链可变区和SEQ ID NO:24的轻链可变区，
    21)SEQ ID NO:23的重链可变区和SEQ ID NO:25的轻链可变区，
    22)SEQ ID NO:23的重链可变区和SEQ ID NO:26的轻链可变区，
    23)SEQ ID NO:23的重链可变区和SEQ ID NO:27的轻链可变区，和
    24)SEQ ID NO:18的重链可变区和SEQ ID NO:24的轻链可变区。
  21. 如权利要求9-16任一项所述的PCSK9抗体或其抗原结合片段，其中所述PCSK9抗体的重链进一步包含人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，或与其具有至少95％序列同一性的氨基酸序列；优选包含人源IgG1、IgG2或IgG4或使用氨基酸突变延长抗体在血清中的半衰期的IgG1、IgG2或IgG4变体的重链恒定区，更优选包含引入YTE突变的IgG1、IgG2或IgG4重链恒定区；
    其中所述PCSK9抗体的轻链进一步包含人源κ、λ链或其变体的恒定区，或与其具有至少95％序列同一性的氨基酸序列。
  22. 根据权利要求21所述的PCSK9抗体或其抗原结合片段，其中所述人源化抗体包含选自以下的重链和轻链：
    1)SEQ ID NO:28的重链和SEQ ID NO:30的轻链，和
    2)SEQ ID NO:32的重链和SEQ ID NO:30的轻链。
  23. 一种药物组合物，其含有治疗有效量的根据权利要求1至22任一项所述的PCSK9抗体或其抗原结合片段，以及一种或多种药学上可接受的载体、稀释剂或赋形剂。
  24. 一种编码根据权利要求1-22任一项所述的PCSK9抗体或其抗原结合片段的DNA分子。
  25. 一种含有根据权利要求24所述的DNA分子的表达载体。
  26. 一种用根据权利要求25所述的表达载体转化的宿主细胞，所述宿主细胞选自原核细胞和真核细胞，优选为真核细胞，更优选哺乳动物细胞。
  27. 如权利要求1至22任一项所述的PCSK9抗体或其抗原结合片段、或如权利要求23所述的药物组合物，在制备用于治疗PCSK9介导的疾病或病症的药物中的用途，其中所述的疾病或病症优选为胆固醇相关疾病；更优选为高胆固醇血症、心脏病、代谢综合征、糖尿病、冠状动脉心脏病、卒中、心血管疾病、阿尔茨海默病和一般性的异常脂血症；最优选高胆固醇血症、异常脂血症、动脉粥样硬化、CVD或冠状动脉心脏病。
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WO2019001560A1 (zh) * 2017-06-30 2019-01-03 江苏恒瑞医药股份有限公司 一种pcsk-9抗体药物组合物及其用途
WO2021185344A1 (zh) 2020-03-19 2021-09-23 江苏恒瑞医药股份有限公司 一种胆固醇相关疾病的治疗方法

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