WO2017071513A1 - 抗人pcsk9单克隆抗体及其用途 - Google Patents

抗人pcsk9单克隆抗体及其用途 Download PDF

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WO2017071513A1
WO2017071513A1 PCT/CN2016/102656 CN2016102656W WO2017071513A1 WO 2017071513 A1 WO2017071513 A1 WO 2017071513A1 CN 2016102656 W CN2016102656 W CN 2016102656W WO 2017071513 A1 WO2017071513 A1 WO 2017071513A1
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sequence
antibody
variable region
chain variable
pcsk9
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PCT/CN2016/102656
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French (fr)
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刘志刚
刘玉兰
郭晶晶
郝小勃
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北京智仁美博生物科技有限公司
智翔（上海）医药科技有限公司
重庆智翔金泰生物制药有限公司
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Publication of WO2017071513A1 publication Critical patent/WO2017071513A1/zh

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    • 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
    • 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

Definitions

  • the present application relates generally to the field of genetic engineering and antibody drugs, and more particularly to monoclonal antibodies against human PCSK9 and uses thereof.
  • the application provides human-derived functional anti-human PCSK9 monoclonal antibodies, and the use of such antibodies in the treatment of human PCSK9 mediated diseases.
  • Cardiovascular disease is currently the leading cause of death in China.
  • Heart disease, stroke and peripheral arterial disease caused by atherosclerosis are the leading causes of death, while elevated levels of low-density lipoprotein cholesterol (LDL-C) are arteries.
  • LDL-C low-density lipoprotein cholesterol
  • Statins are currently the most effective drugs for lowering LDL-C, but some patients still do not reach normal levels of blood lipids after taking the drug, and some patients cannot tolerate statins. Therefore, there is a need to find a potent cholesterol-lowering drug other than statins to reduce the risk of atherosclerotic cardiovascular disease.
  • PCSK9 Proprotein convertase subtilisin kexin 9, PCSK9, also known as neural apoptosis-regulated convertase 1 (NARC-1).
  • PCSK9 protein is mainly synthesized and secreted in the liver.
  • PCSK9 protein enters the blood circulation and binds to the low-density lipoprotein receptor (LDLR) in the liver to mediate LDLR degradation, thereby reducing liver to LDL-C.
  • LDLR low-density lipoprotein receptor
  • PCSK9 knockout mice exhibited approximately a 50% reduction in plasma cholesterol levels and increased sensitivity of statins to lower plasma cholesterol (Rashid S, et al 2005, Proc Nath Acad Sci 102: 5374-5379).
  • the anti-PCSK9 monoclonal antibody can effectively prevent PCSK9 from binding to LDL-R, thereby exerting a powerful cholesterol-lowering effect, and can be used for statin treatment of patients with hypercholesterolemia who are poorly responsive or statin intolerant.
  • PCSK9 inhibitor Repatha (Evolocumab) developed by Amgen and the PCSK9 inhibitor Praluent (Alirocumab) developed by Sanofi/Regeneration have been approved by the European and US FDA for the treatment of primary hypercholesterolemia. Mixed hyperlipidemia and familial hypercholesterolemia.
  • PCSK9 inhibitor Repatha (Evolocumab) developed by Amgen
  • PCSK9 inhibitor Praluent (Alirocumab) developed by Sanofi/Regeneration
  • One of the objects of the present application is to provide a novel monoclonal antibody against human PCSK9 or a fragment thereof.
  • the sequence of the variable region of the antibody gene enables the construction of a full-length antibody molecule as a drug for the clinical treatment of a disease mediated by human PCSK9.
  • Suitable indications include, but are not limited to, the following: primary hypercholesterolemia, mixed hyperlipidemia, and familial hypercholesterolemia.
  • the inventors of the present application screened and optimized the human-derived anti-PCSK9 single from the previously constructed large-capacity natural human phage antibody library (for details, see Chinese Patent Application No. 201510097117.0, the entire contents of which is incorporated herein by reference) Cloning antibodies.
  • the application provides an anti-PCSK9 monoclonal antibody comprising a heavy chain variable region, the heavy chain variable region comprising three HCDRs, characterized in that the HCDR1 sequence is GYX 1 X 2 X 3 NY, and the HCDR2 sequence is SFYNGN, HCDR3 sequence is GYVMDX 4 , wherein X 1 X 2 X 3 is AHI, TLN or ALY, and X 4 is I or F; wherein the HCDR sequence is defined according to Chothia.
  • the anti-PCSK9 monoclonal antibody comprises a heavy chain variable region, and the heavy chain variable region has an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-19.
  • the application provides an anti-PCSK9 monoclonal antibody comprising a light chain variable region, the light chain variable region comprising three LCDRs, wherein the LCDR1 sequence is RASQSINNWLD, the LCDR2 sequence is AASTRPS, and the LCDR3 sequence is QQDQDIPPT; where the LCDR sequence is defined according to Chothia.
  • the anti-PCSK9 monoclonal antibody comprises a light chain variable region, and the light chain variable region has the amino acid sequence of SEQ ID NO:20.
  • the application provides an anti-PCSK9 monoclonal antibody comprising (i) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 sequences, characterized in that the HCDR1 sequence is GYX 1 X 2 X 3 NY, HCDR2 The sequence is SFYNGN, and the HCDR3 sequence is GYVMDX 4 , wherein X 1 X 2 X 3 is AHI, TLN or ALY, X 4 is I or F, wherein the HCDR sequence is defined according to Chothia; and (ii) contains LCDR1, LCDR2 and LCDR3 sequences.
  • the light chain variable region is characterized in that the LCDR1 sequence is RASQSINNWLD, the LCDR2 sequence is AASTRPS, and the LCDR3 sequence is QQDQDIPPT, wherein the LCDR sequence is defined according to Chothia.
  • the heavy chain variable region sequence of the anti-PCSK9 monoclonal antibody is SEQ ID NO:16 and the light chain variable region sequence is SEQ ID NO:20. In some embodiments, the heavy chain variable region sequence is SEQ ID NO:17 and the light chain variable region sequence is SEQ ID NO:20. In some embodiments, the heavy chain variable region sequence is SEQ ID NO: 18 and the light chain variable region sequence is SEQ ID NO: 20. In some embodiments, the heavy chain variable region sequence is SEQ ID NO: 19 and the light chain variable region sequence is SEQ ID NO: 20.
  • an anti-PCSK9 monoclonal antibody can comprise or consist of an intact antibody (ie, a full length antibody), a substantially intact antibody, or an antigen binding portion thereof, eg, a Fab fragment, a F(ab') 2 fragment, or a single strand Fv fragment composition.
  • the anti-PCSK9 monoclonal antibody comprises a constant region.
  • the antibody is heavy
  • the chain constant region can be an IgGl subtype (SEQ ID NO: 7), an IgG2 (SEQ ID NO: 8) or an IgG4 subtype (SEQ ID NO: 9).
  • the light chain constant region can be a kappa subtype (SEQ ID NO: 10) or a lambda subtype (SEQ ID NO: 11).
  • the monoclonal antibody or fragment thereof is of all human origin.
  • the affinity of the antibodies of the present application can be tested according to standard test methods.
  • the measurement can be performed by coupling the antibody against the human F C segment to the surface of the chip CM5 using the instrument Biacore 3000, diluting the antibody protein to a suitable concentration, and ensuring that about 200 RU of the antibody is captured by the antibody against human Fc. .
  • the antibody was subjected to a series of concentration gradients (for example, 100 nm, 50 nm, 25 nm, 12.5 nm, 6.25 nm, 3.125 nm, 1.5625 nm, 0 nm) through the surface of the stationary phase, and the affinity of each monoclonal antibody was determined.
  • the anti-PCSK9 mAb of the present application is effective to inhibit binding of LDLR to PCSK9.
  • the stability of the antibodies of the present application in human serum can be tested according to standard test methods.
  • a sample of the monoclonal antibody to be sterilized can be taken, diluted in 200 ⁇ l of sterile normal human mixed serum or PBS to a final concentration of 30 ⁇ g/ml, mixed, and placed in a 37 ° C water bath for 12 days (288 hours). After 12 days, serum samples (A: antibody/normal human serum), PBS samples (B: antibody/PBS) and frozen monoclonal antibody samples (C: antibodies) were combined with human PCSK9 (huPCSK9) by ELISA, The changes in the ability of each monoclonal antibody to bind huPCSK9 (A/C) were compared.
  • the anti-PCSK9 mAb of the present application has good serum stability.
  • the activity of the antibodies of the present application to inhibit PCSK9-induced down-regulation of cell surface LDLR can be tested according to standard test methods.
  • the HEK293 cell density can be adjusted to 8*10 5 cells/ml and seeded in 24-well plates (500 ⁇ l/well). Then, the antibody to be tested and PCSK9 were added to the cells, and cultured in suspension for 3 hours; then, the cells in each well were separately collected, washed twice with 1% BSA-PBS, and then with FITC-anti-LDLR monoclonal antibody (Sino Biological Inc.
  • the anti-PCSK9 mAb of the present application (at 20 ⁇ g/ml) is capable of significantly inhibiting PCSK9-induced downregulation of LDLR levels.
  • the activity of the antibodies of the present application to inhibit PCSK9-induced cellular uptake of LDL-C can be tested according to standard test methods.
  • the HEK293 cell density can be adjusted to 8*10 5 cells/ml and seeded in 24-well plates (500 ⁇ l/well). Then, PCSK9 and antibody were added to the cells, and cultured in suspension for 2.5 h; then BODIPY FL LDL (Invitrogen, Cat#: L3483) was added to each well to a final concentration of 6 ⁇ g/ml, and suspension culture was continued for 2.5 h; The cells in each well were collected and washed twice with PBS.
  • the BODIPY signal (excitation wavelength 488 nM, emission wavelength 520) was analyzed by BC Accur C6 flow cytometry; untreated HEK293 cells were used as controls, and various treatments were compared. The cells take up the level of LDL-C.
  • the anti-PCSK9 mAb of the present application (at 20 ⁇ g/ml) is capable of significantly inhibiting PCSK9-induced downregulation of LDK-C uptake by HEK293 cells.
  • the antibodies of the present application may be fully human monoclonal antibodies that antagonize at least one in vitro or in vivo biological activity associated with PCSK9 or a portion thereof.
  • an antibody of the present application is effective to inhibit PCSK9 binding to the EGF-AB domain of the recombinant human LDLR extracellular domain.
  • the antibodies of the present application have strong binding affinity to human PCSK9, and the affinity (eg, H7B12-IgG4) is superior or identical to the similar antibody Evolocumab from Amgen and the similar antibody Alirobumab from Sanofi.
  • the application provides the use of a monoclonal antibody of any of the preceding aspects for the treatment of hypercholesterolemia mediated by human PCSK9.
  • the hypercholesterolemia is primary hypercholesterolemia, mixed hyperlipidemia, or familial hypercholesterolemia.
  • the application provides a pharmaceutical composition comprising a monoclonal antibody of any of the foregoing aspects and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further comprises an excipient, a diluent, and the like.
  • the pharmaceutical composition may further comprise a lubricant such as talc, magnesium stearate and mineral oil; a wetting agent; an emulsifier; a suspending agent; a preservative such as benzoic acid, sorbic acid and calcium propionate.
  • a lubricant such as talc, magnesium stearate and mineral oil
  • a wetting agent such as talc, magnesium stearate and mineral oil
  • an emulsifier such as talc, magnesium stearate and mineral oil
  • a suspending agent such as benzoic acid, sorbic acid and calcium propionate.
  • a preservative such as benzoic acid, sorbic acid and calcium propionate.
  • the pharmaceutical compositions of the present application can be formulated in the form of tablets, pills, powders, lozenges, elixirs, suspensions, emulsions, solutions, syrups, suppositories or capsules.
  • the pharmaceutical compositions of the present application can be delivered using any physiologically acceptable administration, including but not limited to: oral administration, parenteral administration, nasal administration, rectal administration. Drug, intraperitoneal administration, intravascular injection, subcutaneous administration, transdermal administration, inhalation administration, and the like.
  • a pharmaceutical composition for therapeutic use may be formulated in a lyophilized formulation or as an aqueous solution by mixing an agent having the desired purity with a pharmaceutically acceptable carrier, excipient, or the like, as appropriate. storage.
  • compositions of the present application are used to treat hypercholesterolemia mediated by human PCSK9.
  • the hypercholesterolemia is primary hypercholesterolemia, mixed hyperlipidemia, or familial Hypercholesterolemia.
  • the application provides a method of treating hypercholesterolemia mediated by human PCSK9 comprising administering to a subject in need thereof a monoclonal antibody or pharmaceutical composition of any of the preceding aspects.
  • the hypercholesterolemia is primary hypercholesterolemia, mixed hyperlipidemia, or familial hypercholesterolemia.
  • Figure 1 shows the results of different monoclonal antibodies inhibiting the binding of PCSK9 to the EGF-AB domain of the extracellular domain of human LDLR.
  • Figure 2 shows the results of a test in which different anti-human PCSK9 monoclonal antibodies compete with LDLR for binding to human PCSK9.
  • Figure 3 shows the results of stability analysis of different anti-huPCSK9 mAbs in human serum.
  • Figure 4 shows that different anti-PCSK9 mAbs inhibit PCSK9-induced down-regulation of LDLR on HEK293 cells (in which anti-IL17 is an anti-IL17 monoclonal antibody; Aliro is anti-PCSK9 control monoclonal antibody Alirobumab; Evolo is an anti-PCSK9 control antibody Evolocumab ; H4H4, H7B12, H8A7, H8F4 are four exemplary anti-PCSK9 mAbs of the present application).
  • Figure 5 shows that different anti-PCSK9 mAbs inhibit PCSK9-induced down-regulation of LDL-C uptake by HEK293 cells (where anti-IL17 is an anti-IL17 monoclonal antibody; Aliro is an anti-PCSK9 control monoclonal antibody Alirocumab; Evolo is anti-PCSK9
  • the control antibodies Evolocumab; H4H4, H7B12, H8A7, H8F4 are four exemplary anti-PCSK9 monoclonal antibodies of the present application).
  • Figure 6 shows the analysis of the binding properties of different anti-human PCSK9 mAbs to different species of PCSK9 by ELISA experiments.
  • Figure 7 is a graph showing the trend of the relative percentage change of the average LDL-C of the Syrian golden hamster.
  • Example 1 Preparation of recombinant protein.
  • PCSK9 human PCSK9
  • mouse PCSK9 mouse PCSK9
  • maPCSK9 maPCSK9, SEQ ID NO: 25
  • cynomolgus PCSK9 mmPCSK9, SEQ ID NO: 3
  • human LDLR extracellular domain EGF-AB, SEQ ID NO: 4
  • a non-antibody recombinant protein has a His tag (SEQ ID NO: 5) or an Fc portion of a murine antibody (mFc, SEQ ID NO: 6) at the C-terminus.
  • the antibody heavy chain constant region may be an IgG1 subtype (SEQ ID NO: 7), an IgG2 subtype (SEQ ID NO: 8) or an IgG4 subtype (SEQ ID NO: 9), and the light chain constant region may Is a kappa subtype (SEQ ID NO: 10) or a Lambda subtype (SEQ ID NO: 11).
  • the genes of the above various recombinant proteins were designed and synthesized based on the amino acid sequence of the recombinant protein for various purposes of the Uniprot database.
  • the recombinant recombinant protein gene is cloned into a suitable eukaryotic expression vector (such as invitrogen pcDNA3.1, etc.) by conventional molecular biology techniques, and then subjected to liposome (such as invitrogen 293fectin, etc.) or other transfer.
  • the prepared recombinant protein expression plasmid is transfected into HEK293 cells (such as HEK293F of Invitrogen) by a staining reagent (such as PEI, etc.), and cultured in serum-free suspension culture conditions for 3-4 days. The culture supernatant is then harvested by centrifugation or the like.
  • HEK293 cells such as HEK293F of Invitrogen
  • a staining reagent such as PEI, etc.
  • the recombinant protein expressed by the His-tag fusion is subjected to one-step purification of the recombinant protein in the culture supernatant by a metal chelate affinity chromatography column (such as GE's HisTrap FF).
  • the recombinant protein and recombinant antibody expressed by mFc fusion were subjected to one-step purification using a ProteinA/G affinity chromatography column (e.g., Mabselect SURE, GE).
  • the recombinant protein storage buffer is then replaced with PBS (pH 7.0) or other suitable buffer using a desalting column (eg, Hitrap desaulting, GE, etc.). If necessary, the antibody samples can be sterilized by filtration and then stored separately at -20 °C.
  • Example 2 Screening and optimization of anti-human PCSK9 mAb using phage display antibody library technology.
  • PCSK9-His The prepared recombinant huPCSK9-his (hereinafter abbreviated as PCSK9-His) was used as an antigen, and the phage displaying the human single-chain antibody library was screened by a solid phase screening strategy to obtain a plurality of different but specific sequences.
  • Human antibodies that bind human PCSK9 including clones: S2B8 (SEQ ID NO: 12), S2F5 (SEQ ID NO: 13), S2G1 (SEQ ID NO: 14), S3A12 (SEQ ID NO: 15). It was found through testing that recombinant whole antibody S3A12 was able to effectively inhibit PCSK9 binding to the EGF-AB domain of the recombinant human LDLR extracellular domain (Fig. 1).
  • Table 1 Affinity matured monoclonal antibodies against human PCSK9.
  • Example 3 BIacore 3000 determines the affinity of whole antibodies.
  • An amine coupling kit and a human antibody capture kit as well as a CM5 chip and 10 x HBS-EP at pH 7.4 were purchased from GE Healthcare.
  • the antibody against the human F C segment was conjugated to the surface of the chip CM5 according to the instructions in the kit, and the antibody protein was diluted to a suitable concentration to ensure that about 200 RU of the antibody was captured by the antibody against human Fc.
  • the huPCSK9 was subjected to a series of concentration gradients (100 nm, 50 nm, 25 nm, 12.5 nm, 6.25 nm, 3.125 nm, 1.5625 nm, 0 nm) through the surface of the stationary phase, and the affinity of each monoclonal antibody was measured, and Evolocumab was measured in the same manner.
  • VH SEQ ID NO: 21, VL: SEQ ID NO: 22
  • Alirocumab VH: SEQ ID NO: 23, VL: SEQ ID NO: 24
  • Example 4 Anti-human PCSK9 monoclonal antibody competes for PCSK9 receptor LDLR binding to PCSK9.
  • Functional anti-PCSK9 mAb should block the binding between LDLR and PCSK9 at the protein level.
  • This example analyzed six different anti-PCSK9 mAbs (H4H4, H7B12, H8A7, H8F4, Evolocumab and Alirocumab) to inhibit LDLR. The ability to combine with PCSK9.
  • Each monoclonal antibody was diluted with 5.6 ⁇ g/ml of PCSK9-his in a total of 11 dilutions. The first 6 gradients were diluted 1:1, and the next 5 gradients were diluted 1:2. The initial concentration of each antibody was adjusted to 30 ⁇ g. /ml.
  • the binding signal of LDLR to PCSK9 was detected by HRP-mouse-anti-his IgG monoclonal antibody, and then data analysis and mapping were performed using GraphPad Prism 6, and the results are shown in Fig. 2.
  • the four antibodies prepared and two commercial control antibodies blocked the binding between LDLR and PCSK9.
  • Example 5 Stability analysis of anti-PCSK9 mAb in human serum.
  • Table 3 Changes in the ability of monoclonal antibody samples to bind huPCSK9 under different treatment conditions.
  • Example 6 Anti-PCSK9 mAb inhibits PCSK9-induced downregulation of LDLR receptors on HEK293 cell surface.
  • HEK293 cells cultured in 293 free medium were used.
  • the HEK293 cell density was adjusted to 8*10 5 cells/ml, and seeded in a 24-well plate (500 ⁇ l/well).
  • the cells were then treated as listed in Table 4 and cultured in suspension for 3 h.
  • the cells in each well were separately collected, washed twice with 1% BSA-PBS, and then incubated with FITC-anti-LDLR mAb (Sino Biological Inc. Cat #10231-R301-F) for 45 minutes on ice and washed with PBS 2 After that, the FITC signal was analyzed using BC's Accuri C6 flow cytometer.
  • Untreated HEK293 cells were used as controls to compare the levels of LDLR on the cell surface after various treatments.
  • the results (Fig. 4) showed that treatment of HEK293 cells with 10 ⁇ g/ml of PCSK9 resulted in a significant decrease in LDLR levels, whereas various anti-PCSK9 mAbs (20 ⁇ g/ml) significantly inhibited PCSK9-induced downregulation of LDLR levels.
  • Table 4 Different treatments for HEK293 cells.
  • Example 7 Anti-PCSK9 mAb inhibits down-regulation of PCSK9-induced ability of HEK293 cells to uptake LDL-C.
  • HEK293 cells cultured in 293 free medium were used.
  • the HEK293 cell density was adjusted to 8*10 5 cells/ml, and seeded in a 24-well plate (500 ⁇ l/well).
  • the cells were then treated as listed in Table 5 and cultured in suspension for 2.5 h.
  • BODIPY FL LDL (Invitrogen, Cat#: L3483) was added to each well to a final concentration of 6 ⁇ g/ml, and suspension culture was continued for 2.5 hours.
  • Table 5 Different treatments for HEK293 cells.
  • Example 8 Binding analysis of anti-PCSK9 mAb with different species of PCSK9.
  • the prepared human PCSK9 (huPCSK9), mouse PCSK9 (moPCSK9), macaque PCSK9 (mmPCSK9) and Syrian golden hamster PCSK9 (maPCSK9) were respectively coated in a 96-well ELISA plate, 4 ⁇ g/ml, 100 ⁇ l/well, 4 ° C package. Was stayed overnight. After blocking with a blocking solution (2% milk-PBST) for 1 hour at 37 ° C, various anti-PCSK9 mAbs were added and combined at 37 ° C for 1 hour. The ELISA plate was washed with PBST, and HRP-anti-human IgG (secondary antibody) was added and conjugated at 37 ° C for 1 hour.
  • a blocking solution 2% milk-PBST
  • the ELISA plate was washed with PBST, and the OPD substrate coloring solution was added. After 5-10 minutes, the color development was stopped with 1 M H 2 SO 4 , and the optical density value was measured at 490 nm by a microplate reader.
  • the anti-PCSK9 mAb and the control antibody prepared in the present application were able to bind human, macaque and Syrian golden hamster PCSK9, but did not bind to mouse PCSK9.
  • the anti-PCSK9 mAb of the present application is capable of recognizing the Syrian golden hamster PCSK9 (maPCSK9), so this example uses the Syrian golden hamster (Gold Syrian hamster) for two exemplary anti-PCSK9 mAbs (H7B12). And H8A7) in vivo hypolipidemic studies.
  • a random grouping method was used to give each eligible animal a random number using Excel software. The animals were sorted into small groups according to the order of random numbers, and each group of 6 animals. Animals were fed a normal diet, and each group of animals received a single subcutaneous administration of the monoclonal antibody at a dose of 15 mg/kg.
  • All animals were administered three days prior to dosing (D-3) and the second day after dosing (D2), fourth day (D4), seventh day (D7), tenth day (D10), and fourteenth day ( D14) and the 21st day (D21) blood collection and separation of serum, total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL- C) Determination.
  • TC total cholesterol
  • TG triglyceride
  • LDL-C low density lipoprotein cholesterol
  • HDL- C high density lipoprotein cholesterol

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Abstract

提供了利用噬菌体抗体库技术筛选并利用基因工程方法制备的抗人PCSK9的单克隆抗体或其片段，还提供了所述单克隆抗体或其片段在治疗由人PCSK9介导的疾病中的用途。

Description

抗人PCSK9单克隆抗体及其用途 技术领域
本申请大体涉及基因工程和抗体药物领域，更具体地涉及抗人PCSK9的单克隆抗体及其应用。本申请提供了人源性功能性抗人PCSK9单克隆抗体，以及这类抗体在治疗人PCSK9介导的疾病中的用途。
背景技术
心血管疾病是目前中国首位的死亡原因，其中动脉粥样硬化引起的心脏病、脑卒中和外周动脉疾病是导致死亡的主要原因，而低密度脂蛋白胆固醇(LDL-C)水平升高是动脉粥样硬化性心血管疾病最大的危险因素。他汀类药物是目前降低LDL-C最有效的药物，但是仍有部分患者在服用药物后血脂水平不能达到正常水平，并且有些患者不能耐受他汀类药物。因此，需要寻找他汀以外的强效降胆固醇药物，以降低动脉粥样硬化性心血管疾病的风险的方法。
前蛋白枯草溶菌素转化酶9(proprotein convertase subtilisin kexin 9,PCSK9)，又称为神经凋亡调节转化酶1(neural apoptosis-regulated convertase 1,NARC-1)。PCSK9蛋白主要在肝脏中合成和分泌，PCSK9蛋白进入血液循环后，在肝脏与低密度脂蛋白受体(the low-density lipoprotein receptor,LDLR)结合，介导LDLR降解，从而降低肝脏对LDL-C的清除能力，升高血浆LDL-C水平。PCSK9基因敲除的小鼠表现出血浆胆固醇水平大约降低50％，而且使他汀类药物降低血浆胆固醇的灵敏度变高(Rashid S,et al 2005,Proc Nath Acad Sci 102:5374-5379)。
抗PCSK9单克隆抗体可有效阻止PCSK9与LDL-R结合，从而发挥强效降胆固醇的作用，可用于他汀治疗响应不良或他汀不耐受的高胆固醇血症的患者。
目前安进研发的PCSK9抑制剂Repatha(Evolocumab)和赛诺菲/再生元研发的PCSK9抑制剂Praluent(Alirocumab)都已经获得欧洲和美国FDA的批准上市，用于治疗原发性高胆固醇血症、混合型高脂血症和家族性高胆固醇血症。但是本领域仍然需要更多适于治疗患者的改进的抗PCSK9抗体。
发明内容
本申请的目的之一在于提供新型抗人PCSK9的单克隆抗体或其片段。由本申请涉及 的抗体基因可变区的序列，可构建全长的抗体分子作为药物，用于临床上由人PCSK9介导的疾病的治疗。适合的适应症包括但不局限于下列：原发性高胆固醇血症、混合型高脂血症和家族性高胆固醇血症。
本申请的发明人从先前构建的大容量天然人源噬菌体抗体库(具体内容参见中国专利申请201510097117.0号，通过引用将该申请的全部内容并入本文)中筛选并优化得到了人源抗PCSK9单克隆抗体。
在一个方面，本申请提供了抗PCSK9单克隆抗体，其含有重链可变区，重链可变区含有3个HCDR，其特征在于，HCDR1序列为GYX1X2X3NY，HCDR2序列为SFYNGN，HCDR3序列为GYVMDX4，其中X1X2X3为AIY、TLN或者ALY，X4为I或者F；其中HCDR序列根据Chothia定义。在一些实施方案中，抗PCSK9单克隆抗体包含重链可变区，重链可变区具有选自SEQ ID NO:16-19的氨基酸序列。
在另一方面，本申请提供了抗PCSK9单克隆抗体，其含有轻链可变区，轻链可变区含有3个LCDR，其特征在于，LCDR1序列为RASQSINNWLD，LCDR2序列为AASTRPS，LCDR3序列为QQDQDIPPT；其中LCDR序列根据Chothia定义。在一些实施方案中，抗PCSK9单克隆抗体包含轻链可变区，轻链可变区具有SEQ ID NO:20的氨基酸序列。
在另一方面，本申请提供了抗PCSK9单克隆抗体，其含有(i)含HCDR1、HCDR2和HCDR3序列的重链可变区，其特征在于，HCDR1序列为GYX1X2X3NY，HCDR2序列为SFYNGN，HCDR3序列为GYVMDX4，其中X1X2X3为AIY、TLN或者ALY，X4为I或者F，其中HCDR序列根据Chothia定义；以及(ii)含LCDR1、LCDR2和LCDR3序列的轻链可变区，其特征在于，LCDR1序列为RASQSINNWLD，LCDR2序列为AASTRPS，LCDR3序列为QQDQDIPPT，其中LCDR序列根据Chothia定义。
在一些实施方案中，抗PCSK9单克隆抗体的重链可变区序列为SEQ ID NO：16，轻链可变区序列为SEQ ID NO：20。在一些实施方案中，重链可变区序列为SEQ ID NO：17，轻链可变区序列为SEQ ID NO：20。在一些实施方案中，重链可变区序列为SEQ ID NO：18，轻链可变区序列为SEQ ID NO：20。在一些实施方案中，重链可变区序列为SEQ ID NO：19，轻链可变区序列为SEQ ID NO：20。
在一些实施方案中，抗PCSK9单克隆抗体可包含或由完整的抗体(即全长抗体)、基本上完整的抗体或其抗原结合部分，例如Fab片段、F(ab')2片段或单链Fv片段组成。
在一些实施方案中，抗PCSK9单克隆抗体包括恒定区。在一些实施方案中，抗体重 链恒定区可以是IgG1亚型(SEQ ID NO:7)、IgG2(SEQ ID NO:8)或者IgG4亚型(SEQ ID NO:9)。在一些实施方案中，轻链恒定区可以是kappa亚型(SEQ ID NO:10)或者lambda亚型(SEQ ID NO:11)。
在一些实施方案中，单克隆抗体或其片段是全人源的。
本申请的抗体的亲和力可以按照标准测试方法进行测试。作为非限制性的实例，测量可使用仪器Biacore 3000，将抗人FC段的抗体偶联至芯片CM5的表面上，稀释抗体蛋白至合适浓度，保证200RU左右的抗体被抗人Fc的抗体捕获。将抗体设置一系列的浓度梯度(例如，100nm，50nm，25nm，12.5nm，6.25nm，3.125nm，1.5625nm，0nm)流经固定相表面，测定各单克隆抗体的亲和力。
在一些实施方案中，本申请的抗PCSK9单抗能有效抑制LDLR与PCSK9的结合。
本申请的抗体在人血清中的稳定性可以按照标准测试方法进行测试。作为非限制性的实例，可以取过滤去菌的单抗样品，分别稀释于200μl无菌的正常人混合血清或PBS至终浓度30μg/ml，混匀后置37℃水浴放置12天(288小时)；12天后，利用ELISA分析血清样品(A:抗体/正常人血清)，PBS样品(B:抗体/PBS)和冻存的单抗样品(C:抗体)与人PCSK9(huPCSK9)的结合，并分别比较各单抗样品结合huPCSK9能力的变化(A/C)。
在一些实施方案中，本申请的抗PCSK9单抗具有好的血清稳定性。
本申请的抗体的抑制PCSK9诱导细胞表面LDLR下调的活性可以按照标准测试方法进行测试。作为非限制性的实例，可以将HEK293细胞密度调整至8*105细胞/ml，接种于24孔板(500μl/孔)。然后在细胞中加入待测抗体和PCSK9，悬浮培养3h；然后分别收集各孔中细胞，用1％BSA-PBS洗涤2次，然后与FITC-抗LDLR单抗(Sino Biological Inc.Cat#10231-R301-F)于冰上孵育45分钟，用PBS洗涤2次后，用BD公司的Accuri C6流式细胞仪分析FITC信号；以未处理的HEK293细胞为对照，比较各种处理后细胞表面LDLR的水平。
在一些实施方案中，本申请的抗PCSK9单抗(以20μg/ml)能够明显抑制PCSK9诱导的LDLR水平下调。
本申请的抗体抑制PCSK9诱导细胞摄取LDL-C能力的活性可以按照标准测试方法进行测试。作为非限制性的实例，可以将HEK293细胞密度调整至8*105细胞/ml，接种于24孔板(500μl/孔)。然后在细胞中加入PCSK9和抗体，悬浮培养2.5h；然后再分别在每个孔中补加BODIPY FL LDL(Invitrogen，Cat#:L3483)至终浓度6μg/ml，继续悬浮培养2.5h； 然后分别收集各孔中细胞，用PBS洗涤2次后，用BD公司的Accuri C6流式细胞仪分析BODIPY信号(激发波长488nM，发射波长520)；以未处理的HEK293细胞为对照，比较各种处理后细胞摄取LDL-C的水平。
在一些实施方案中，本申请的抗PCSK9单抗(以20μg/ml)能够明显抑制PCSK9诱导的HEK293细胞摄取LDL-C水平的下调。
本申请的抗体可以为全人源的单克隆抗体，其能拮抗至少一种与PCSK9或其部分相关的体外或体内生物活性。
在一些实施方案中，本申请的抗体能够有效抑制PCSK9结合重组人LDLR胞外区EGF-AB结构域。
在一些实施方案中，本申请的抗体对人PCSK9具有强结合亲和力，亲和力(例如，H7B12-IgG4)优于或与安进公司的同类抗体Evolocumab以及赛诺菲公司的同类抗体Alirocumab相同。
在另一方面，本申请提供了前文任一方面的单克隆抗体在治疗由人PCSK9介导的高胆固醇血症中的用途。
在一些实施方案中，高胆固醇血症为原发性高胆固醇血症、混合型高脂血症或家族性高胆固醇血症。
在一些实施方案中，本申请提供了药物组合物，其包含前文任一方面的单克隆抗体和药学可接受的载体。
在一些实施方案中，药物组合物还包含赋形剂、稀释剂等。
在一些实施方案中，药物组合物还可包含润滑剂，如滑石粉、硬脂酸镁和矿物油；润湿剂；乳化剂；悬浮剂；防腐剂，如苯甲酸、山梨酸和丙酸钙；增甜剂和/或调味剂等。
在一些实施方案中，可将本申请中的药物组合物配制为片剂、丸剂、粉剂、锭剂、酏剂、悬液、乳剂、溶液、糖浆、栓剂或胶囊等形式。
在一些实施方案中，可以利用任何生理上可接受的给药方式递送本申请的药物组合物，这些给药方式包括但不限于：口服给药、肠胃外给药、经鼻给药、直肠给药、腹膜内给药、血管内注射、皮下给药、经皮给药、吸入给药等。
在一些实施方案中，可以通过混合具有所需纯度的试剂与视情况的药学上可接受的载体、赋形剂等，以冻干制剂或水溶液的形式配制用于治疗用途的药物组合物用于存储。
在一些实施方案中，本申请的药物组合物用于治疗由人PCSK9介导的高胆固醇血症。
在一些实施方案中，高胆固醇血症为原发性高胆固醇血症、混合型高脂血症或家族性 高胆固醇血症。
在另一方面，本申请提供了治疗由人PCSK9介导的高胆固醇血症的方法，包括向有需要的个体给予前文任一方面的单克隆抗体或药物组合物。
在一些实施方案中，高胆固醇血症为原发性高胆固醇血症、混合型高脂血症或家族性高胆固醇血症。
附图说明
图1显示了不同单抗抑制PCSK9结合人LDLR胞外区EGF-AB结构域的测试结果。
图2显示了不同抗人PCSK9单克隆抗体与LDLR竞争结合人PCSK9的测试结果。
图3显示了不同抗huPCSK9单抗在在人血清中的稳定性分析结果。
图4显示了不同抗PCSK9单抗抑制PCSK9诱导的HEK293细胞表面的LDLR下调(其中抗IL17为一株抗IL17的单抗；Aliro为抗PCSK9的对照单抗Alirocumab；Evolo为抗PCSK9的对照抗体Evolocumab；H4H4，H7B12，H8A7，H8F4为本申请的四株示例性抗PCSK9单抗)。
图5显示了不同抗PCSK9单抗抑制PCSK9诱导的HEK293细胞摄取LDL-C能力的下调(其中抗IL17为一株抗IL17的单抗；Aliro为抗PCSK9的对照单抗Alirocumab；Evolo为抗PCSK9的对照抗体Evolocumab；H4H4，H7B12，H8A7，H8F4为本申请的四株示例性抗PCSK9的单抗)。
图6显示了通过ELISA实验分析不同抗人PCSK9单抗对不同种属PCSK9结合特性。
图7显示了叙利亚金黄地鼠平均LDL-C相对百分比变化趋势图。
实施例
以下实施例仅仅对本发明进行进一步的说明，不应理解为对本发明的限制。
实施例1：重组蛋白的制备。
制备和筛选抗PCSK9单抗的过程中用到多种不同的重组蛋白，包括人PCSK9(huPCSK9，SEQ ID NO:1),小鼠PCSK9(moPCSK9,SEQ ID NO:2)，叙利亚金黄地鼠PCSK9(maPCSK9，SEQ ID NO:25)和猕猴PCSK9(mmPCSK9,SEQ ID NO:3)，人LDLR胞外区(EGF-AB，SEQ ID NO:4)，以及重组抗体。这些蛋白都有大量的翻译后修饰(如：糖基化或二硫键等)，因而利用哺乳动物细胞表达系统将更有利于保持重组蛋白的结构和功 能。此外，为了方便纯化，非抗体类的重组蛋白在C端添加了His标签(SEQ ID NO:5)或者鼠抗体的Fc段(mFc，SEQ ID NO:6)。重组抗体制备时，抗体重链恒定区可以是IgG1亚型(SEQ ID NO:7)，IgG2亚型(SEQ ID NO:8)或者IgG4亚型(SEQ ID NO:9)，轻链恒定区可以是kappa亚型(SEQ ID NO:10)或者Lambda亚型(SEQ ID NO:11)。
根据Uniprot数据库的各种目的重组蛋白的氨基酸序列，设计并合成上述各种重组蛋白的基因(包含His-tag或者mFc编码基因)。利用常规的分子生物学技术将合成的各种重组蛋白基因克隆至合适的真核表达载体(如invitrogen公司的pcDNA3.1等)，然后利用脂质体(如invitrogen公司的293fectin等)或其它转染试剂(如PEI等)将制备的重组蛋白表达质粒转染入HEK293细胞(如invitrogen公司的HEK293F)，在无血清悬浮培养条件下培养3-4天。然后通过离心等方式收获培养上清。
His标签融合表达的重组蛋白利用金属螯合亲和层析柱(如GE公司的HisTrap FF等)对培养上清中的重组蛋白进行一步纯化。而mFc融合表达的重组蛋白和重组抗体用ProteinA/G亲和层析柱(如GE公司的Mabselect SURE等)进行一步纯化。然后利用脱盐柱(如GE公司的Hitrap desaulting等)将重组蛋白保存缓冲液置换为PBS(pH7.0)或者其它合适的缓冲液。必要时，可以对抗体样品进行过滤除菌，然后分装保存于-20℃。
实施例2：利用噬菌体呈现抗体库技术筛选和优化抗人PCSK9单抗。
以制备的重组huPCSK9-his(以下简写为PCSK9-His)为抗原，参照文献(中国专利申请201510097117.0号)，利用固相筛选策略筛选呈现人单链抗体库的噬菌体，获得多株序列不同但特异性结合人PCSK9的人抗体，包括克隆：S2B8(SEQ ID NO:12)，S2F5(SEQ ID NO:13)，S2G1(SEQ ID NO:14)，S3A12(SEQ ID NO:15)。通过测试发现，重组全抗体S3A12能够有效抑制PCSK9结合重组人LDLR胞外区EGF-AB结构域(图1)。
然后参照文献(中国专利申请201510097117.0号)利用轻链置换和重链CDR突变的策略对抗体S3A12进行体外亲和力成熟，最终获得四株亲和力提高的抗人PCSK9的单抗，具体序列信息见表1。
表1：亲和力成熟的抗人PCSK9的单抗。
Figure PCTCN2016102656-appb-000001
实施例3：BIacore3000测定全抗体的亲和力。
胺偶联试剂盒和人抗体捕获试剂盒以及CM5芯片和pH7.4的10×HBS-EP均购自GE Healthcare。
根据试剂盒中的说明书，将抗人FC段的抗体偶联至芯片CM5的表面上，稀释抗体蛋白至合适浓度，保证200RU左右的抗体被抗人Fc的抗体捕获。将huPCSK9设置一系列的浓度梯度(100nm，50nm，25nm，12.5nm，6.25nm，3.125nm，1.5625nm，0nm)流经固定相表面，测定各单克隆抗体的亲和力，用同样的方法测定Evolocumab(VH：SEQ ID NO:21，VL：SEQ ID NO:22)和Alirocumab(VH：SEQ ID NO:23，VL：SEQ ID NO:24)的亲和力作为对照，结果如表2所示。所制备的四株抗体均具有理性的结合亲和力，并且其中H7B12的结合亲和力还优于商业化对照抗体Evolocumab和Alirocumab。
表2.抗huPCSK9单抗亲和力常数测定数值。
抗体 Kon(1/MS) Koff(1/S) KD
H4H4-IgG4 7.14E+05 4.70E-05 65.9pM
H7B12-IgG4 1.01E+06 3.26E-05 32.3pM
H8F4-IgG4 6.63E+05 4.48E-05 67.5pM
H8A7-IgG4 7.46E+05 4.83E-05 64.8pM
Evolocumab-IgG4 2.28E+05 1.93E-05 76.4pM
Alirocumab 2.27E+06 1.54E-04 67.7pM
实施例4：抗人PCSK9单克隆抗体竞争PCSK9受体LDLR结合PCSK9。
功能性的抗PCSK9单抗应该能在蛋白水平上阻断LDLR和PCSK9之间的结合，本实施例分析了6种不同抗PCSK9单抗(H4H4、H7B12、H8A7、H8F4、Evolocumab和Alirocumab)抑制LDLR与PCSK9结合的能力。
用5.6μg/ml的PCSK9-his对各单克隆抗体进行梯度稀释，共11个稀释浓度，前6个梯度1:1稀释，后5个梯度1:2稀释，各抗体起始浓度调整为30μg/ml。利用HRP-小鼠-抗his IgG单抗检测LDLR与PCSK9的结合信号，然后利用GraphPad Prism 6进行数据分析和作图，结果如图2所示。所制备的四株抗体以及两株商业化对照抗体均能阻断LDLR和PCSK9之间的结合。
实施例5：抗PCSK9单抗在人血清中的稳定性分析。
为了分析不同抗PCSK9单抗分子的特异性及血清稳定性，进行了抗PCSK9单抗在人血清中的稳定性分析。此研究包括6种不同抗PCSK9单抗，分别为H4H4，H7B12，H8A7，H8F4，Evolocumab和Alirocumab。取过滤去菌的单抗样品，分别稀释于200μl无菌的正常 人混合血清或PBS至终浓度30μg/ml，混匀后置37℃水浴放置12天(288小时)。12天后，利用ELISA分析血清样品(A:抗体/正常人血清)，PBS样品(B:抗体/PBS)和冻存的单抗样品(C:抗体)与huPCSK9的结合，并分别比较各单抗样品结合huPCSK9能力的变化(A/C)。结果(表3以及图3)表明本发明中的四株抗huPCSK9单抗具有好的血清稳定性。
表3：不同处理条件下单抗样品结合huPCSK9能力的变化。
Figure PCTCN2016102656-appb-000002
实施例6：抗PCSK9单抗抑制PCSK9诱导HEK293细胞表面LDLR受体的下调。
本实施例选用悬浮培养于293自由培养基(Invitrogen)的HEK293细胞。将HEK293细胞密度调整至8*105细胞/ml，接种于24孔板(500μl/孔)。然后按照表4所列方式处理细胞，悬浮培养3h。然后分别收集各孔中细胞，用1％BSA-PBS洗涤2次，然后与FITC-抗LDLR单抗(Sino Biological Inc.Cat#10231-R301-F)于冰上孵育45分钟，用PBS洗涤2次后，用BD公司的Accuri C6流式细胞仪分析FITC信号。以未处理的HEK293细胞为对照，比较各种处理后细胞表面LDLR的水平。结果(图4)显示：10μg/ml的PCSK9处理HEK293细胞后导致细胞表明LDLR水平明显下降，而各种抗PCSK9单抗(20μg/ml)能够明显抑制PCSK9诱导的LDLR水平下调。
表4：HEK293细胞的不同处理方式。
Figure PCTCN2016102656-appb-000003
实施例7：抗PCSK9单抗抑制PCSK9诱导HEK293细胞摄取LDL-C能力的下调。
本实施例选用悬浮培养于293自由培养基(Invitrogen)的HEK293细胞。将HEK293细胞密度调整至8*105细胞/ml，接种于24孔板(500μl/孔)。然后按照表5所列方式处理细胞，悬浮培养2.5h。然后再分别在每个孔中补加BODIPY FL LDL(Invitrogen，Cat#:L3483)至终浓度6μg/ml，继续悬浮培养2.5h。然后分别收集各孔中细胞，用PBS洗涤2次后，用BD公司的Accuri C6流式细胞仪分析BODIPY信号(激发波长488nM，发射波长520)。以未处理的HEK293细胞为对照，比较各种处理后细胞摄取LDL-C的水平。结果(图5)显示：10μg/ml的PCSK9处理HEK293细胞后导致细胞摄取LDL-C量明显下降，而各种抗PCSK9单抗(20μg/ml)能够明显抑制PCSK9诱导的HEK293细胞摄取LDL-C水平的下调。
表5：HEK293细胞的不同处理方式。
Figure PCTCN2016102656-appb-000004
实施例8：抗PCSK9单抗与不同种属PCSK9结合分析。
将制备的人PCSK9(huPCSK9)、小鼠PCSK9(moPCSK9)、猕猴PCSK9(mmPCSK9)和叙利亚金黄地鼠PCSK9(maPCSK9)分别包被于96孔ELISA板，4μg/ml，100μl/孔，4℃包被过夜。利用封闭液(2％牛乳-PBST)在37℃封闭1小时后，分别加入各种抗PCSK9单抗，37℃结合1小时。用PBST洗涤ELISA板，加入HRP-抗人IgG(二抗)，37℃ 结合1小时。PBST洗涤ELISA板，加入OPD底物显色液，5-10分钟后用1M H2SO4终止显色，酶标仪490nm测定光密度值。结果如图6所示，本申请制备的抗PCSK9单抗及对照抗体能够结合人、猕猴和叙利亚金黄地鼠PCSK9，但不结合小鼠PCSK9。
实施例9：抗PCSK9单抗下调正常叙利亚金黄地鼠的LDL-C
如前文实施例所证实，本申请的抗PCSK9单抗能够识别叙利亚金黄地鼠PCSK9(maPCSK9)，因此本实施例选用叙利亚金黄地鼠(Gold Syrian hamster)进行两种示例性抗PCSK9单抗(H7B12和H8A7)体内降血脂研究。采用随机分组法，用Excel软件给予每只合格的动物一个随机数，按照随机数从小到大的顺序排序，将动物分为3组，每组6只动物。动物都进行正常饮食喂养，各组动物都进行单抗药物的单次皮下给药，剂量为15mg/kg。所有动物在给药前三天(D-3)和给药后第二天(D2)，第四天(D4)，第七天(D7)，第十天(D10)，第十四天(D14)和第二十一天(D21)采血并分离血清，分别进行总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)测定。结果(图7)显示H7B12和H8A7两种单抗的给药分别能够将血清中LDL-C浓度降低57％和41％。

Claims (17)

  1. 一种特异性结合人PCSK9的单克隆抗体，其包含含HCDR1、HCDR2和HCDR3序列的重链可变区，其特征在于，所述HCDR1序列为GYX1X2X3NY，所述HCDR2序列为SFYNGN，所述HCDR3序列为GYVMDX4，其中X1X2X3为AIY、TLN或者ALY，X4为I或者F；其中HCDR序列根据Chothia定义。
  2. 根据权利要求1所述的单克隆抗体，其特征在于，所述抗体的重链可变区序列如SEQ ID NO：16、17、18或19所示。
  3. 一种特异性结合人PCSK9的单克隆抗体，其包含含LCDR1、LCDR2和LCDR3序列的轻链可变区，其特征在于，所述LCDR1序列为RASQSINNWLD，所述LCDR2序列为AASTRPS，所述LCDR3序列为QQDQDIPPT；其中LCDR序列根据Chothia定义。
  4. 根据权利要求3所述的单克隆抗体，其特征在于，所述抗体的轻链可变区序列如SEQ ID NO：20所示。
  5. 一种特异性结合PCSK9的单克隆抗体，其包含含HCDR1、HCDR2和HCDR3序列的重链可变区及含LCDR1、LCDR2和LCDR3序列的轻链可变区，其特征在于，所述HCDR1序列为GYX1X2X3NY，所述HCDR2序列为SFYNGN，所述HCDR3序列为GYVMDX4，所述LCDR1序列为RASQSINNWLD，所述LCDR2序列为AASTRPS，所述LCDR3序列为QQDQDIPPT，其中X1X2X3为AIY、TLN或者ALY，X4为I或者F；其中HCDR和LCDR序列根据Chothia定义。
  6. 根据权利要求5所述的单克隆抗体，其中
    所述抗体的重链可变区序列为SEQ ID NO：16，轻链可变区序列为SEQ ID NO：20；或
    重链可变区序列为SEQ ID NO：17，轻链可变区序列为SEQ ID NO：20；或
    重链可变区序列为SEQ ID NO：18，轻链可变区序列为SEQ ID NO：20；或
    重链可变区序列为SEQ ID NO：19，轻链可变区序列为SEQ ID NO：20。
  7. 根据权利要求1至6中任何一项的抗体，其中所述抗体为全长抗体、基本上完整的抗体、Fab片段、F(ab')2片段或单链Fv片段。
  8. 根据权利要求7所述的单克隆抗体，其特征在于，所述抗体是全人源抗体。
  9. 根据权利要求1至6中任何一项的抗体，其中所述抗体包含选自IgG1、IgG2或IgG4亚型的重链恒定区和/或包含选自kappa或lambda亚型的轻链恒定区。
  10. 根据权利要求1至6中任何一项的抗体，其用作药物。
  11. 权利要求1-10中任一项的单克隆抗体在治疗由人PCSK9介导的高胆固醇血症中的用途。
  12. 根据权利要求11所述的用途，其特征在于，所述高胆固醇血症为原发性高胆固醇血症、混合型高脂血症或家族性高胆固醇血症。
  13. 药物组合物，其包含权利要求1-10中任一项的单克隆抗体和药学可接受的载体。
  14. 根据权利要求13所述的药物组合物，其用于治疗由人PCSK9介导的高胆固醇血症。
  15. 根据权利要求14所述的药物组合物，所述高胆固醇血症为原发性高胆固醇血症、混合型高脂血症或家族性高胆固醇血症。
  16. 治疗由人PCSK9介导的高胆固醇血症的方法，包括向有需要的个体给予权利要求1-10中任一项的单克隆抗体或权利要求13-15中任一项的药物组合物。
  17. 根据权利要求16所述的方法，所述高胆固醇血症为原发性高胆固醇血症、混合型高脂血症或家族性高胆固醇血症。
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