WO2018133649A1 - 抗pcsk9单克隆抗体 - Google Patents

抗pcsk9单克隆抗体 Download PDF

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WO2018133649A1
WO2018133649A1 PCT/CN2017/119822 CN2017119822W WO2018133649A1 WO 2018133649 A1 WO2018133649 A1 WO 2018133649A1 CN 2017119822 W CN2017119822 W CN 2017119822W WO 2018133649 A1 WO2018133649 A1 WO 2018133649A1
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seq
dfsk9
antibody
pcsk9
heavy chain
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PCT/CN2017/119822
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English (en)
French (fr)
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周海平
李晓敏
张稳
文圣梅
白义
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北京东方百泰生物科技有限公司
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Priority to JP2019505510A priority Critical patent/JP6734467B2/ja
Priority to AU2017394444A priority patent/AU2017394444B2/en
Priority to CA3032506A priority patent/CA3032506C/en
Priority to EP17893120.0A priority patent/EP3480216A4/en
Priority to US16/321,472 priority patent/US11390688B2/en
Priority to KR1020197003329A priority patent/KR102257462B1/ko
Priority to EA201990170A priority patent/EA039191B1/ru
Publication of WO2018133649A1 publication Critical patent/WO2018133649A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
    • 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
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • 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 the field of antibody engineering technology, and in particular relates to a monoclonal antibody against PCSK9 which is fully human, and a method and application thereof.
  • PCSK9 Protein convertase subtilisin/kexin type 9 belongs to the preprotein invertase family proteinase K subfamily.
  • the human PCSK9 gene is located on chromosome 1p32.3 and is approximately 22 kb in length with a total of 12 exons encoding a protein of 692 amino acid residues.
  • the PCSK9 protein consists of a signal peptide, a pro-domain, a catalytic domain and a carboxy-terminal domain (V domain).
  • PCSK 9 is mainly expressed in the liver, intestines and kidneys, and is also expressed in small amounts in the skin and nervous system, but only PCSK9 in the liver can be secreted into the blood circulatory system.
  • PCSK9 can mediate low-density lipoprotein receptor (LDLR) degradation and regulate plasma LDL cholesterol (LDL-C) levels, while LDLR-mediated intrahepatic LDL endocytosis is the primary removal of LDL from the circulatory system.
  • LDLR is a multi-domain protein, and the extracellular domain is closely linked to the EGF-A, EGF-B, and EGF-C of the epidermal growth factor precursor.
  • PCSK9-mediated LDLR degradation first needs to bind to LDLR.
  • the binding site on LDLR is mainly EGF-A, forming a complex of PCSK9 and EGF-A.
  • PCSK9 can also regulate cholesterol metabolism through very low-density lipoprotein receptor, apolipoprotein B receptor, and apolipoprotein E receptor 2, but the molecular mechanism is not clear.
  • inhibitors of PCSK9 mainly include monoclonal antibodies, antisense nucleotides, small interfering RNAs, peptidomimetics and small molecule inhibitors.
  • Monoclonal antibody drugs are the research and development hotspots in the field of biomedicine this year. They are characterized by strong targeting, high specificity and low toxic and side effects, and represent the latest development direction in the field of drug therapy. Monoclonal antibodies targeting PCSK9 can specifically bind to PCSK9, block the interaction between PCSK9 and LDLR, slow down the degradation process of LDLR and play a role in lowering LDL-C levels. Clinical trial data show the safety, efficacy and unique clinical application value of anti-PCSK9 monoclonal antibody.
  • Fully human antibodies are the main direction of the development of therapeutic antibodies.
  • the emergence of antibody library technology provides a good technical platform for the preparation and screening of human antibodies.
  • the antibody library technology bypasses the hybridoma process necessary in the development of the monoclonal antibody in the past, and even obtains various antibody genes and antibody molecular fragments without the need of an immunological process.
  • the phage antibody library is the earliest and currently the most widely used antibody library.
  • the phage antibody library is divided into an immunological library and a non-immune library according to the source of the antibody gene, and the non-immune library includes a natural library, a semi-synthetic library, and a fully synthetic library.
  • Screening of phage antibody libraries mimics the process of antibody affinity maturation, usually by coating the antigen on a solid phase medium, adding the phage antibody library to be screened, and through several rounds of "adsorption-wash-elution-amplification” processes (ie Panning) until screening for high affinity specific antibodies.
  • Evololcumab is well tolerated and there are currently no obvious safety issues.
  • Pfizer's humanized monoclonal antibody bococizumab is undergoing Phase III clinical trials, and Novartis's humanized monoclonal antibody lodelcizumab is undergoing Phase II clinical trials.
  • Roche and Merck are also conducting clinical trials.
  • the invention provides a monoclonal antibody against PCSK9; the invention selects a monoclonal antibody against PCSK9 from a fully synthetic antibody library, and then constructs a small-capacity synthetic phage antibody light chain library by computer-aided design analysis, and obtains a screening method
  • the CDR1, 2, and 3 regions of the light chain of the anti-PCSK9 monoclonal antibody were constructed. After screening, the monoclonal antibody with higher affinity was selected, and the CDR1, 2, and 3 regions of the heavy chain were screened for finalization.
  • a high affinity monoclonal antibody against PCSK9 was screened.
  • the anti-PCSK9 antibody of the invention has a novel sequence, and the antibody functions well in vitro, especially at the cellular level, and has great medical application prospects.
  • the present invention relates to a monoclonal antibody against PCSK9, comprising:
  • LCDR1 comprises RASQSIDNRLT (SEQ NO. 22), RASQSVRNWLD (SEQ NO. 23), RASQGINSWLN (SEQ NO. 24), RASQNVNNWLN (SEQ NO. 25), RASQNINSWLN (SEQ NO. 26), RASQNINNWLN (SEQ NO. 27) , RASQGIHNWLN (SEQ NO.
  • LCDR2 comprises DASSRQS ( SEQ NO. 33), GASTLES (SEQ NO. 34), AASTRET (SEQ NO. 35), GASSRQS (SEQ NO. 36), GASTRPT (SEQ NO. 37), DASNRQS (SEQ NO. 38), GASNLAS (SEQ NO .39), any of DASNLQS (SEQ NO. 40) or DASSRPT (SEQ NO. 41); LCDR3 comprises QQPENDPTT (SEQ NO.
  • HCDR1 comprises any one of GGTFTNNA (SEQ NO. 53), GYTVTSYG (SEQ NO. 54) or GYSLTSYG (SEQ NO.
  • HCDR2 comprises RIIPMFGMA (SEQ NO. 56) , WLSFYNGNT (SEQ NO. 57), WVTFYNGNT (SEQ NO. 58), WVSFYQGNT (SEQ NO. 59), WVSFYNGQT (SEQ NO. 60) or WVSFYNGNS (SEQ NO. 61);
  • HCDR3 comprises AREGRIPMI ( SEQ NO. 62), ARGYSLDV (SEQ NO. 63), ARGYGMSI (SEQ NO. 64), ARGFGMDR (SEQ NO. 65), ARGYGMTV (SEQ NO. 66) or ARGFGLSV (SEQ NO. 67).
  • amino acid sequence of the light chain variable region is preferably SEQ NO. 11, SEQ NO. 12, SEQ NO. 13, SEQ NO. 14, SEQ NO. 15, SEQ NO. 16, SEQ NO. 17, SEQ NO. 18. Any one of SEQ NO. 19, SEQ NO. 20 or SEQ NO. 21.
  • amino acid sequence of the heavy chain variable region is preferably SEQ NO. 1, SEQ NO. 2, SEQ NO. 3, SEQ NO. 4, SEQ NO. 5, SEQ NO. 6, SEQ NO. 7, SEQ NO. 8. Any one of SEQ NO. 9 or SEQ NO.
  • the heavy chain variable region HCDR1 sequence is an amino acid sequence selected from the group consisting of GYTVTSYG (SEQ NO. 54) or GYSLTSYG (SEQ NO. 55);
  • the light chain variable region LCDR1 sequence is selected from the group consisting of RASQSVRNWLD (SEQ NO. 23) , any one of RASQNVNNWLN (SEQ NO. 25), RASQNINSWLN (SEQ NO. 26), RASQNINNWLN (SEQ NO. 27) or RASQDVDSWLT (SEQ NO. 29);
  • the heavy chain variable region HCDR2 sequence An amino acid sequence selected from the group consisting of WVSFYQGNT (SEQ NO. 59), WVSFYNGQT (SEQ NO.
  • the light chain variable region LCDR2 sequence is selected from GASTLES (SEQ NO. 34) Any one of amino acid sequences of AASTRET (SEQ NO. 35), GASSRQS (SEQ NO. 36), GASTRPT (SEQ NO. 37) or GASNLAS (SEQ NO. 39); said heavy chain variable region HCDR3 sequence An amino acid sequence selected from the group consisting of ARGYSLDV (SEQ NO. 63), ARGYGMSI (SEQ NO. 64), ARGFGMDR (SEQ NO. 65) or ARGYGMTV (SEQ NO.
  • the light chain variable region LCDR3 sequence Any one of amino acid sequences selected from the group consisting of QQDNDIPLT (SEQ NO. 43), QQDNDMPLT (SEQ NO. 45), QQWFDVPTT (SEQ NO. 46), QQWDDTPNT (SEQ NO. 47), or QQDSKIPLT (SEQ NO. 49).
  • the present invention also provides various antibodies, polypeptides or proteins comprising the above light chain or the above heavy chain.
  • the present invention also provides various antibodies comprising the above light chain or the above heavy chain, which can specifically bind to PCSK9, block the binding of PCSK9 to LDLR, increase the number of LDLR on the cell surface or LDLR in the blood circulation system. The level of LDL or LDL-C in the blood circulation system is lowered.
  • the present invention also provides various polynucleotide sequences or combinations comprising the above light chain or the above heavy chain.
  • said anti-PCSK9 heavy chain constant region comprises a monoclonal antibody IgG1, IgG2, IgG3 and lgG4; comprising a light chain constant region C ⁇ or C ⁇ .
  • the heavy chain constant region is preferably IgG4 or IgG2; the eukaryotic expression vector or prokaryotic expression vector of the heavy chain.
  • the light chain constant region is preferably C ⁇ ; the eukaryotic expression vector or prokaryotic expression of the light chain
  • the present invention also provides a recombinant DNA expression vector comprising the above polynucleotide sequence or combination; the DNA sequence of the recombinant DNA expression vector comprises the above heavy chain variable region and heavy chain constant region encoding an anti-PCSK9 antibody Amino acid sequences of the light chain variable region and the light chain constant region.
  • the present invention also provides a host cell transfected with the above recombinant DNA expression vector, the host cell comprising a prokaryotic cell, a yeast, an insect or a mammalian cell.
  • the prokaryotic cell is preferably Escherichia coli.
  • the mammalian cells are preferably HEK293E cells, CHO cells or NSO cells.
  • the present invention provides a plurality of full length antibodies, single chain antibodies, single domain antibodies, bispecific antibodies, and antibody drug conjugates comprising the above light chain or the above heavy chain.
  • the present invention also provides various monoclonal antibodies, artificial carriers, drugs or pharmaceutical compositions containing the above light chain or the above heavy chain.
  • the monoclonal antibody comprises a full length antibody and a fragment of an anti-PCSK9 monoclonal antibody, including but not limited to Fab, Fab', F(ab') 2 , Fv or ScFv.
  • the present invention also provides a detection reagent or kit comprising the above light chain or the above heavy chain.
  • the antibodies of the present invention are useful for diseases which are ameliorated, alleviated, inhibited or prevented by eliminating, inhibiting or reducing PCSK9 activity; the diseases include dyslipidemia, cardiovascular and cerebrovascular diseases, and thromboembolic diseases.
  • the dyslipidemia includes elevated cholesterol, elevated triglycerides, elevated low-density lipoprotein, and decreased high-density lipoprotein.
  • the cardiovascular disease includes coronary heart disease, acute myocardial infarction, atherosclerosis, stroke, and peripheral arterial occlusive disease.
  • the method for obtaining a monoclonal antibody against PCSK9 of the present invention comprises the following steps:
  • DFSK9-16, DFSK9-17, DFSK9-18, DFSK9-19, DFSK9-20 and DFSK9-21 among them, DFSK9-12, DFSK9-13, DFSK9-14, DFSK9-15, DFSK9-16, DFSK9-17
  • the light chain variable region sequences of DFSK9-18, DFSK9-19, DFSK9-20 and DFSK9-21 are DFSK9-L2, DFSK9-L8, DFSK9-L5, DFSK9-L6, DFSK9-L5, DFSK9-L6, DFSK9, respectively.
  • SK9-H8 DFSK9-H7, DFSK9-H3, DFSK9-H6, DFSK9-H4, DFSK9-H4, DFSK9-H9, DFSK9-H5, DFSK9-H10; the corresponding amino acid sequences are shown in SEQ NO. 2, SEQ NO, respectively. .8, SEQ NO. 7, SEQ NO. 3, SEQ NO. 6, SEQ NO. 4, SEQ NO. 4, SEQ NO. 9, SEQ NO. 5, SEQ NO. 10; the above single-chain antibody in phage Affinity comparison at the level;
  • the monoclonal heavy chain variable region gene and the light chain variable gene described in (3) are cloned into a eukaryotic expression vector, and transfected into a host cell to obtain a total anti-PCSK9 monoclonal antibody antibody.
  • the CDR is a complementarity-determining region;
  • the ScFv is a single-chain fragment variable;
  • the ADCs are antibody-drug conjugates;
  • the LDLR a low density lipoprotein receptor (Low Density Lipoprotein Receptor);
  • the LDL-C is Low Density Lipoprotein-Cholesterol;
  • the HEK293E cell is a human embryonic kidney 293E cell;
  • the CHO cells are Chinese hamster ovary cells;
  • the NS0 cells are mouse NSO thymoma cells.
  • the invention has the following beneficial effects:
  • the invention provides a plurality of novel anti-PCSK9 antibodies, which have high affinity for binding to a substrate, can well block the binding of PCSK9 to LDLR, and affect the distribution and expression of LDLR on the cell surface, thereby increasing the LDL on the cell surface.
  • the ability of -R to bind to LDL increases the uptake and degradation of LDL by cells, and reduces the levels of extracellular LDL and LDL-C to reduce LDL, LDL-C and total cholesterol.
  • the monoclonal antibody provided by the present invention can be used for diseases which are improved, alleviated, inhibited or prevented by eliminating, inhibiting or reducing PCSK9 activity; the diseases include dyslipidemia, cardiovascular and cerebrovascular diseases, and thromboangiopathy; dyslipidemia contains cholesterol Elevation, elevated triglycerides, elevated low-density lipoprotein, and decreased high-density lipoprotein; cardiovascular disease includes coronary heart disease, acute myocardial infarction, atherosclerosis, stroke, and peripheral arterial occlusive disease Wait.
  • the present invention provides a monoclonal antibody that specifically binds to PCSK9, the heavy chain variable region sequence comprising any one of SEQ NO. 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.
  • the light chain variable region sequence comprises any one of SEQ NO. 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and 21.
  • the heavy chain variable region sequence of the monoclonal antibody that specifically binds to PCSK9 is selected from any one of SEQ NO. 2, 3, 4, 5, 6, 7, 8, 9, and 10.
  • the light chain variable region sequence is selected from any one of SEQ NO. 12, 14, 15, 16 and 18.
  • amino acid sequences of the light chain complementarity determining regions LCDR1, LCDR2 and LCDR3 of the antibody light chain or a functional fragment thereof are selected from any combination of the following amino acid sequences (as shown in Table 1) by light chain phage library screening:
  • HCDR1 is GGTFTNNA (SEQ NO. 53), GYTVTSYG (SEQ) Any one of NO.54) or GYSLTSYG (SEQ NO. 55);
  • HCDR2 is RIIPMFGMA (SEQ NO. 56), WLSFYNGNT (SEQ NO. 57), WVTFYNGNT (SEQ NO. 58), WVSFYQGNT (SEQ NO. 59) Any one of WVSFYNGQT (SEQ NO. 60) or WVSFYNGNS (SEQ NO.
  • HCDR3 is AREGIPMI (SEQ NO. 62), ARGYSLDV (SEQ NO. 63), ARGYGMSI (SEQ NO. 64), ARGFGMDR (SEQ NO. 65), any of ARGYGMTV (SEQ NO. 66) or ARGFGLSV (SEQ NO. 67).
  • the monoclonal antibody that specifically binds to PCSK9 comprises a heavy chain variable region of the HCDR1, HCDR2 and HCDR3 sequences and a light chain variable region comprising the LCDR1, LCDR2 and LCDR3 sequences, by screening through a heavy chain phage library;
  • the heavy chain variable region HCDR1 sequence is an amino acid sequence selected from the group consisting of GYTVTSYG (SEQ NO. 54) or GYSLTSYG (SEQ NO. 55);
  • the light chain variable region LCDR1 sequence is selected from the group consisting of RASQSVRNWLD (SEQ NO. 23), An amino acid sequence of any one of RASQNVNNWLN (SEQ NO. 25), RASQNINSWLN (SEQ NO.
  • the heavy chain variable region HCDR2 sequence is selected from An amino acid sequence of any one of WVSFYQGNT (SEQ NO. 59), WVSFYNGQT (SEQ NO. 60) or WVSFYNGNS (SEQ NO. 61);
  • the light chain variable region LCDR2 sequence is selected from GASTLES (SEQ NO. 34), An amino acid sequence of AASTRET (SEQ NO. 35), GASSRQS (SEQ NO. 36), GASTRPT (SEQ NO. 37) or GASNLAS (SEQ NO.
  • the heavy chain variable region HCDR3 sequence is selected from the group consisting of ARGYSLDV (SEQ NO. 63), ARGYGMSI (SEQ NO. 64), ARGFGMDR (SEQ NO. 65) or ARGYGMTV (SEQ NO. 66) amino acid sequence;
  • the light chain variable region The LCDR3 sequence is selected from any one of the amino acid sequences of QQDNDIPLT (SEQ NO. 43), QQDNDMPLT (SEQ NO. 45), QQWFDVPTT (SEQ NO. 46), QQWDDTPNT (SEQ NO. 47), or QQDSKIPLT (SEQ NO. 49).
  • the invention provides a method for obtaining a specific antibody by using a fully synthetic ScFv single-chain phage antibody library, wherein the whole human-derived monoclonal antibody specific for PCSK9 is obtained by screening with a phage antibody library technology, and the steps are as follows:
  • the light chain CDR123 mutation library was constructed by computer-aided design, and the bio-panning and positive clones of the antibody library were screened and identified, and antibody sequences of 10 different light chains were obtained.
  • DFSK9-2, DFSK9-3, DFSK9-4, DFSK9-5, DFSK9-6, DFSK9-7, DFSK9-8, DFSK9-9, DFSK9-10, DFSK9-11 were cloned. Affinity comparison of the above 10 single-chain antibodies at the phage level;
  • the monoclonal heavy chain variable region gene and the light chain variable gene described in (3) are cloned into a eukaryotic expression vector, and transfected into a host cell to obtain a total anti-PCSK9 monoclonal antibody antibody.
  • the total resistance of the anti-PCSK9 monoclonal antibody of (4) is tested for affinity and biological activity.
  • the pCom3 vector was engineered by gene cloning, and the modified vector was named pScFvDisb-S1 (Fig. 1). A fully synthetic phage antibody library was constructed based on this vector.
  • the antigen was coated with antigen PCSK9-His 10 ⁇ g/1 ml/tube and coated overnight at 4 °C.
  • the immunotube and phage antibody libraries were closed with PBST-4%milk (phage input was approximately 10 9 -10 12 ) and blocked at 37 ° C for 1 h.
  • the blocked phage antibody library was added to the immunotube for antigen-antibody binding, and reacted at 37 ° C for 1 h. Unbound phage was washed away with PBST-PBS, eluted with 0.1 M Glycine-HCl, pH 2.2, and neutralized with 1.5 M Tris-HCl, pH 8.8, to pH 7.0.
  • the eluate was infected with 10 ml of XL1-Blue bacteria grown to an OD value of about 0.5-0.8, and allowed to stand at 37 ° C for 30 min, and then shake-cultured at 150 rpm for 1 h. 1% of the bacterial solution was taken out for gradient dilution and plated on a 2YTATG small plate for calculation of phage yield. The remaining bacterial solution was centrifuged, coated on a 2YTATG large plate, and cultured overnight at 37 °C.
  • the overnight culture was transferred to 2YTATG liquid medium, shaken to log phase, M13K07 helper phage infection was added, phage was expanded overnight at 28 °C, and phage was purified by PEG6000-NaCl for the next round of screening. A total of 3 rounds of phage library enrichment screening were performed.
  • the well-separated monoclonal colonies were picked, inoculated into 96-well deep-well plates supplemented with 2YTATG liquid medium, and cultured at 37 ° C, 220 rpm for about 5 h to their logarithmic growth phase, adding about 10 10 per well.
  • the helper phage M13KO7 was allowed to stand at 37 ° C for 30 min, and cultured at 150 rpm for 1 h with shaking. After centrifugation at 4000 rpm for 15 min, the pellet was resuspended in 2YTATKA liquid medium and incubated overnight at 28 ° C, 220 rpm.
  • the single-chain antibody DFSK9-1 with higher affinity was screened, and its heavy chain variable region was named DFSK9-H1, its amino acid sequence is shown in SEQ NO.1; its light chain variable region was named DFSK9-L1, and the amino acid sequence is shown in SEQ. NO.11;
  • SEQ NO. 1 (DFSK9-H1 heavy chain variable region sequence):
  • SEQ NO. 11 (DFSK9-L1 light chain variable region sequence):
  • the pScFvDisb-DFSK9-1 plasmid was digested with NheI and NotI, and the 5.5 kb band was recovered by enzymatic cleavage of the agarose gel.
  • the synthesized light chain mutant library gene VLCDR123M was carried out with NheI and NotI. Double digestion, universal product recovery kit to recover the product.
  • the mutant library gene was ligated with the vector molar ratio of 3:1 for 4 h at 16 °C with T4 DNA ligase.
  • the ligation product was electroporated into the XL1-Blue electroporation competent state. Resuscitation was carried out at 37 ° C, shaking at 150 rpm for 1 h.
  • the constructed antibody library has a storage capacity of about 10 8 , and 20 clones were randomly selected for sequence analysis. The sequence correctness and diversity were greater than 90%.
  • Biopanning and positive clone screening were carried out according to the methods of Example 1 and Example 2.
  • the clones with higher affinity were sequenced, and 10 different single-chain antibody sequences were obtained, which were named DFSK9-2 and DFSK9-3, respectively.
  • SEQ NO. 12 (DFSK9-L2 light chain variable region sequence):
  • SEQ NO. 13 (DFSK9-L3 light chain variable region sequence):
  • SEQ NO. 14 (DFSK9-L4 light chain variable region sequence):
  • SEQ NO. 15 (DFSK9-L5 light chain variable region sequence):
  • SEQ NO. 16 (DFSK9-L6 light chain variable region sequence):
  • SEQ NO. 17 (DFSK9-L7 light chain variable region sequence):
  • SEQ NO. 18 (DFSK9-L8 light chain variable region sequence):
  • SEQ NO. 19 (DFSK9-L9 light chain variable region sequence):
  • SEQ NO. 20 (DFSK9-L10 light chain variable region sequence):
  • SEQ NO. 21 (DFSK9-L11 light chain variable region sequence):
  • the clone obtained in this Example 3.2 was subjected to display and purification of monoclonal phage, and a relative dilution of the single-chain antibody was identified by a gradient dilution ELISA at the phage level.
  • PCSK9-His 300 ng/well/100 ⁇ l was coated with 0.01 M PBS buffer at pH 7.2 and coated overnight at 4 °C.
  • the cells were washed 3 times with PBST, and blocked with PBST-4%milk at 37 ° C for 1 h.
  • a purified phage sample 100 ⁇ l/well) diluted with a PBST-4%milk five-fold gradient was added and allowed to stand at 37 ° C for 1 h.
  • the cells were washed 5 times with PBST, and anti-M13-HRP monoclonal antibody (100 ⁇ l/well) diluted 1:5000 in PBST-4%milk was added, and left at 37 ° C for 1 h.
  • the TMB color development kit was developed (100 ⁇ l/well), developed at room temperature for 10 min, and the color development was stopped with 2M H 2 SO 4 (50 ⁇ l/well).
  • the microplate reader reads 450 nm and 630 nm.
  • the data was analyzed and plotted using the software GraphPad Prism 5Demo.
  • the results are shown in Figure 3. The results showed that the selected phage single-chain antibodies were able to bind to PCSK9, and DFSK9-2, DFSK9-4, DFSK9-5, DFSK9-6
  • the affinity with DFSK9-8 was significantly higher than that of other clones, and the above five single-chain antibodies were selected for the next experiment.
  • the mixed plasmids of five single-chain antibodies, such as DFSK9-2, DFSK9-4, DFSK9-5, DFSK9-6 and DFSK9-8, in Example 3.3 were double-digested with NcoI-HF and KpnI, and the size of the gel was recovered by 5.5 kb.
  • the band of the heavy chain mutant library gene VHCDR123M was double-digested with NcoI-HF and KpnI, and the digestion product was recovered by a universal recovery kit.
  • the mutant library gene was ligated with the vector molar ratio of 3:1 for 4 h at 16 °C with T4 DNA ligase. The ligation product was electroporated into the XL1 electroporation competent state.
  • Resuscitation was carried out at 37 ° C, 150 rpm for 1 h. Take 1% of the bacterial solution, dilute it and apply a small plate to calculate the storage capacity. The remaining bacterial solution was centrifuged at 4000 rpm for 15 min, and the pellet was applied to a 2YTATG large plate and cultured at 37 ° C overnight.
  • the constructed antibody library capacity was about 5*10 8 , and 20 clones were randomly selected for sequence analysis. The correct rate and diversity were more than 90%.
  • Example 4.1 The antibody library of Example 4.1 was subjected to phage display and purification recovery. Single-chain antibodies against PCSK9 were panned from this pool. The biopanning method of the phage antibody library and the screening of the positive clones were the same as in the case of Example 1 and Example 2. After sequencing, a total of 10 different anti-PCSK9 antibody sequences were screened and named as DFSK9-12, DFSK9-13, DFSK9-14, DFSK9-15, DFSK9-16, DFSK9-17, DFSK9-18, DFSK9-19, DFSK9-20, DFSK9-21.
  • the light chain variable region sequence of DFSK9-12 is DFSK9-L2, the corresponding amino acid sequence is shown in SEQ NO.
  • the light chain variable region sequence of DFSK9-13 is DFSK9-L8, and the corresponding amino acid sequence is shown in SEQ NO.
  • the light chain variable region sequence of DFSK9-14, DFSK9-16 and DFSK9-18 is DFSK9-L5, the corresponding amino acid sequence is shown in SEQ NO. 15; DFSK9-15, DFSK9-17, DFSK9-20 and DFSK9-21
  • the light chain variable region sequence is DFSK9-L6, the corresponding amino acid sequence is shown in SEQ NO. 16; the light chain variable region sequence of DFSK9-19 is DFSK9-L4, and the corresponding amino acid sequence is shown in SEQ NO.
  • the heavy chain variable region sequence of DFSK9-12 is DFSK9-H2, the corresponding amino acid sequence is shown in SEQ NO. 2; the heavy chain variable region sequence of DFSK9-13 is DFSK9-H8, and the corresponding amino acid sequence is shown in SEQ NO.
  • the heavy chain variable region sequence of DFSK9-14 is DFSK9-H7, the corresponding amino acid sequence is shown in SEQ NO. 7; the heavy chain variable region sequence of DFSK9-15 is DFSK9-H3, and the corresponding amino acid sequence is shown in SEQ NO.
  • the heavy chain variable region sequence of DFSK9-16 is DFSK9-H6, the corresponding amino acid sequence is shown in SEQ NO.
  • the heavy chain variable region sequence of DFSK9-17 and DFSK9-18 is DFSK9-H4, and the corresponding amino acid sequence is shown in SEQ NO. 4;
  • the heavy chain variable region of DFSK9-19 is DFSK9-H9, the corresponding amino acid sequence is shown in SEQ NO. 9;
  • the heavy chain variable region of DFSK9-20 is DFSK9-H5, and the corresponding amino acid sequence is shown in SEQ NO. .5;
  • the heavy chain variable region sequence of DFSK9-21 is DFSK9-H10, and the corresponding amino acid sequence is shown in SEQ NO.
  • SEQ NO. 2 (DFSK9-H2 heavy chain variable region sequence):
  • SEQ NO. 3 (DFSK9-H3 heavy chain variable region sequence):
  • SEQ NO. 4 (DFSK9-H4 heavy chain variable region sequence):
  • SEQ NO. 5 (DFSK9-H5 heavy chain variable region sequence):
  • SEQ NO. 6 (DFSK9-H6 heavy chain variable region sequence):
  • SEQ NO. 7 (DFSK9-H7 heavy chain variable region sequence):
  • SEQ NO. 8 (DFSK9-H8 heavy chain variable region sequence):
  • SEQ NO. 9 (DFSK9-H9 heavy chain variable region sequence):
  • SEQ NO. 10 (DFSK9-H10 heavy chain variable region sequence):
  • Example 4.2 The clone obtained in this Example 4.2 was subjected to display and purification of a monoclonal phage, and the affinity of the single-chain antibody was identified by a phage gradient dilution ELISA test in the same manner as in Example 3, 3.3. The results are shown in Figure 5.
  • the 10 different single-chain antibodies screened were able to bind well to PCSK9 with higher affinity than the original single-stranded DFSK9-1.
  • the heavy chain VH screened to the antibody in Example 4 was cloned into the vector pTSEG4 (Fig. 6) containing the heavy chain constant region gene (?4), and the light chain VK gene was cloned into a vector containing the light chain constant region gene (kappa chain).
  • pTSEK Fig. 7
  • the vectors pTSEG4 and pTSEK were all modified on the basis of the PTT vector.
  • the preparation of the PTT vector is described in detail in the reference (Yves. Durocher, Sylvie. Perret and Amine. Kamen Nucleic Acids Research, 2002 Vol. 30, No. 2e9).
  • HEK293E cells were transiently transfected for full antibody expression. Whole antibody protein was obtained using an AKTA instrument protein A affinity column purification.
  • PCSK9-His (300 ng/well/100 ⁇ l) was coated with 0.01 M PBS buffer at pH 7.2 and coated overnight at 4 °C. Wash 3 times with 300 ⁇ l/well PBST (1 ⁇ Tween 20), and then add PBST-4%milk for 1 h at 37 °C.
  • the cells were washed 5 times with 300 ⁇ l/well PBST, developed by TMB color development kit (100 ⁇ l/well), developed at room temperature for 10 min, and then stopped to develop color with 2M H 2 SO 4 (50 ⁇ l/well).
  • the microplate reader reads 450 nm and 630 nm.
  • the data was analyzed using the software GraphPad Prism 5Demo. The results of the experiment are shown in Figure 8 and Table 2. All antibodies were well bound to the PCSK9 molecule, including DFSK9-14, DFSK9-15, DFSK9-16, DFSK9-17, DFSK9-18, DFSK9-20 and DFSK9-21 have relatively high affinities.
  • LDLR-Fc (100 ng/well/100 ⁇ l) was coated with 0.01 M PBS, pH 7.2, and coated overnight at 4 °C. The cells were washed 3 times with PBST, and then PBST-4%milk was added at 37 ° C for 1 h.
  • PCSK9-His (100 ⁇ l/well) at a concentration of 2 ⁇ g/ml diluted with PBST-4%milk was added and incubated at 37 ° C for 1 h.
  • PBST-4%milk was added to dilute different dilutions of whole antibody DFSK9-1, DFSK9-12, DFSK9-13, DFSK9-14, DFSK9-15, DFSK9-16, DFSK9-17, DFSK9-18, DFSK9-19, DFSK9 -20 and DFSK9-21.
  • the highest concentration of 11 full antibodies was 100 ug/ml, a five-fold gradient dilution, 8 gradients for each full antibody, and incubation at 37 °C for 2 h.
  • the cells were washed 5 times with PBST, and a mouse anti-His IgG-HRP secondary antibody diluted with PBST-4%milk was added and incubated at 37 ° C for 1 h.
  • the TMB color development kit was developed (100 ⁇ l/well), developed at room temperature for 10 min, and the color development was stopped with 2M H 2 SO 4 (50 ⁇ l/well).
  • the microplate reader reads 450 nm and 630 nm.
  • the affinity of the whole antibody was determined by the capture method.
  • the goat anti-human IgG was coupled to the surface of the CM5 chip, and DFSK9-1, DFSK9-12, DFSK9-13, DFSK9-14, DFSK9-15, DFSK9-16, DFSK9-17, DFSK9-18, DFSK9-19, respectively, were diluted.
  • DFSK9-20 and DFSK9-21 ensure that approximately 200 RU of antibody is captured by goat anti-human IgG.
  • PCSK9 was subjected to a series of concentration gradients (200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.5625 nM, 0.78125 nM) flowing through the surface of the stationary phase to determine the affinity of the antibody.
  • concentration gradients 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.5625 nM, 0.78125 nM
  • HepG2 cells were seeded into 96 wells at 2.5 ⁇ 10 5 cells/ml. The next day, 10% FBS EMEM growth medium was changed to 80 ⁇ l of analysis medium containing 5% delipoprotein FBS, and cultured at 37 ° C for 24 h. On the next day, whole dilutions of the culture medium diluted in the assay medium (10 ⁇ l/well) were added to the culture plates inoculated with HepG2 cells. Eight full-antibody samples were initially spiked at 900 nmol/L in 3-fold gradient dilutions with 8 gradients per full antibody.

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Abstract

抗PCSK9的单克隆抗体、包含编码所述抗体可变区和CDR区的氨基酸序列、所述单克隆抗体的获得方法及用途。从噬菌体抗体库中筛选出抗PCSK9的单克隆抗体，通过链置换构建噬菌体库的方法进行亲和力成熟，先对初筛获得的单克隆抗体的轻链CDR1、2、3区突变建库筛选后，选取亲和力较高的单克隆抗体，再对其重链CDR1、2、3区突变建库进行筛选，最终筛选到了高亲和力的抗PCSK9的单克隆抗体。所述PCSK9抗体对PCSK9具有良好的亲和力并能够抑制PCSK9与其配体的结合，可用于血脂异常、心脑血管疾病和血栓闭塞性疾病的治疗。

Description

抗PCSK9单克隆抗体 技术领域
本发明涉及抗体工程技术领域，具体涉及本发明涉及全人源的抗PCSK9的单克隆抗体、及其获得方法与应用。
背景技术
PCSK9(前蛋白转化酶枯草溶菌素9，Proprotein convertase subtilisin/kexin type 9)属于前蛋白转化酶家族蛋白酶K亚家族。人PCSK9基因定位于染色体1p32.3，长约22kb，共有12个外显子，编码含692个氨基酸残基的蛋白质。PCSK9蛋白由信号肽、前结构域、催化结构域和羧基末端结构域(V结构域)组成，它作为可溶性的74kDa的前体合成，在内质网中经过自身催化裂解产生14kDa的前肽和60kDa的成熟的蛋白酶。PCSK 9主要在肝脏、肠道和肾脏表达，皮肤和神经系统也有少量表达，但是只有肝脏中的PCSK9可以分泌到血液循环系统中。
研究表明，PCSK9能介导低密度脂蛋白受体(LDLR)降解从而调节血浆中LDL胆固醇(LDL-C)水平，而LDLR介导肝内的LDL胞吞过程是从循环系统中清除LDL的主要途径。LDLR是1个多结构域的蛋白，胞外域与表皮生长因子前体同源结构域EGF-A、EGF-B、EGF-C紧连。PCSK9介导LDLR降解首先需要与LDLR结合，LDLR上结合位点主要是EGF-A，形成PCSK9与EGF-A复合物。有研究表明，PCSK9也可通过极低密度脂蛋白受体、载脂蛋白B受体、载脂蛋白E受体2调节胆固醇代谢，但其中的分子机制并不明确。
基础研究和临床试验表明，通过外源性的干预措施抑制PCSK9活性，可以加速血浆中LDL的清除，从而达到降血脂的作用。目前PCSK9的抑制剂主要包括单克隆抗体、反义核苷酸、小分子干扰RNA、模拟肽和小分子抑制剂等。
单克隆抗体药物是今年生物医药领域的研发热点，具有靶向性强、特异性高和毒副作用低等特点，代表了药品治疗领域的最新发展方向。以PCSK9为靶标的单克隆抗体可以与PCSK9发生特异性结合，阻断PCSK9与LDLR的相互作用，减慢LDLR的降解过程从而发挥降低LDL-C水平的作用。临床实验数据显示了抗PCSK9单抗药物的安全性、有效性及独特的临床应用价值。
全人源抗体是治疗性抗体发展的主要方向。抗体库技术的出现为人源抗体的制备筛选提供了良好的技术平台。抗体库技术绕过了以往单抗研制过程中必须的 杂交瘤过程，甚至不需要经过免疫过程即可获得各种抗体基因及抗体分子片段。噬菌体抗体库是最早出现也是目前应用最广泛的抗体库。噬菌体抗体库根据抗体基因的来源分为免疫库和非免疫库，非免疫库又包括天然库、半合成库和全合成库。噬菌体抗体库的筛选模拟了抗体亲和力成熟的过程，通常将抗原包被在固相介质上，加入待筛选的噬菌体抗体库，通过数轮“吸附-洗涤-洗脱-扩增”的过程(即淘选)直至筛选到高亲和力特异的抗体。
目前多家制药公司都在积极开发针对PCSK9的单抗药物。安进的Repatha(evolocumab)和赛诺菲/再生元的Praluent(alirocumab)均为全人源抗体，已于2015年先后被批准上市，用于原发性高胆固醇血症和家族性高胆固醇血症(杂合子和纯合子)的治疗。在他汀基础上加用Evolocumab可使原发性高胆固醇血症患者的LDL-C降低77％，杂合子家族性高胆固醇血症患者的LDL-C降低68％，纯合子家族性高胆固醇血症患者的LDL-C降低31％。evolocumab耐受性良好，目前无任何明显的安全性问题。辉瑞的人源化单抗bococizumab正在进行三期临床，诺华的人源化单抗lodelcizumab正在进行二期临床。罗氏及默沙东也在开展临床试验。
目前国内该领域依然缺乏自主研发的具有高亲和力的抗PCSK9的全人源抗体。
发明内容
本发明提供了抗PCSK9的单克隆抗体；本发明从全合成抗体库中筛选出抗PCSK9的单克隆抗体，然后通过计算机辅助设计分析，构建小容量合成噬菌体抗体轻链库的方法，对筛选获得的抗PCSK9单克隆抗体的轻链的CDR1、2、3区突变建库；筛选后，选取亲和力较高的单克隆抗体，再对其重链CDR1、2、3区突变建库进行筛选，最终筛选到了高亲和力的抗PCSK9的单克隆抗体。本发明抗PCSK9抗体具有全新的序列，该抗体在体外特别是细胞水平上功能良好，极具医学应用前景。
为实现上述目的，本发明一种抗PCSK9的单克隆抗体，包括：
轻链和重链；所述轻链的互补决定区CDR1、CDR2和CDR3分别用LCDR1、LCDR2和LCDR3表示；并且所述重链的互补决定区CDR1、CDR2和CDR3分别用HCDR1、HCDR2和HCDR3表示；LCDR1包含RASQSIDNRLT(SEQ NO.22)、RASQSVRNWLD(SEQ NO.23)、RASQGINSWLN(SEQ NO.24)、RASQNVNNWLN(SEQ NO.25)、RASQNINSWLN(SEQ NO.26)、RASQNINNWLN(SEQ NO.27)、RASQGIHNWLN(SEQ NO.28)、RASQDVDSWLT(SEQ  NO.29)、RASQSVRNWLN(SEQ NO.30)、RASQDVRNWLT(SEQ NO.31)或RASQSIRSYLN(SEQ NO.32)中的任一种；LCDR2包含DASSRQS(SEQ NO.33)、GASTLES(SEQ NO.34)、AASTRET(SEQ NO.35)、GASSRQS(SEQ NO.36)、GASTRPT(SEQ NO.37)、DASNRQS(SEQ NO.38)、GASNLAS(SEQ NO.39)、DASNLQS(SEQ NO.40)或DASSRPT(SEQ NO.41)中的任一种；LCDR3包含QQPENDPTT(SEQ NO.42)、QQDNDIPLT(SEQ NO.43)、QQWNNTPNT(SEQ NO.44)、QQDNDMPLT(SEQ NO.45)、QQWFDVPTT(SEQ NO.46)、QQWDDTPNT(SEQ NO.47)、QQNSNIPLT(SEQ NO.48)、QQDSKIPLT(SEQ NO.49)、QQWTDTPLT(SEQ NO.50)、QQDDSTPPT(SEQ NO.51)或QQGDSMPMT(SEQ NO.52)中的任一种；HCDR1包含GGTFTNNA(SEQ NO.53)、GYTVTSYG(SEQ NO.54)或GYSLTSYG(SEQ NO.55)中的任一种；HCDR2包含RIIPMFGMA(SEQ NO.56)、WLSFYNGNT(SEQ NO.57)、WVTFYNGNT(SEQ NO.58)、WVSFYQGNT(SEQ NO.59)、WVSFYNGQT(SEQ NO.60)或WVSFYNGNS(SEQ NO.61)中的任一种；HCDR3包含AREGIPMI(SEQ NO.62)、ARGYSLDV(SEQ NO.63)、ARGYGMSI(SEQ NO.64)、ARGFGMDR(SEQ NO.65)、ARGYGMTV(SEQ NO.66)或ARGFGLSV(SEQ NO.67)。
其中，所述轻链可变区氨基酸序列优选SEQ NO.11、SEQ NO.12、SEQ NO.13、SEQ NO.14、SEQ NO.15、SEQ NO.16、SEQ NO.17、SEQ NO.18、SEQ NO.19、SEQ NO.20或SEQ NO.21中的任一种。
其中，所述重链可变区氨基酸序列优选SEQ NO.1、SEQ NO.2、SEQ NO.3、SEQ NO.4、SEQ NO.5、SEQ NO.6、SEQ NO.7、SEQ NO.8、SEQ NO.9或SEQ NO.10中的任一种。
其中，所述重链可变区HCDR1序列为选自GYTVTSYG(SEQ NO.54)或GYSLTSYG(SEQ NO.55)的氨基酸序列；所述轻链可变区LCDR1序列选自RASQSVRNWLD(SEQ NO.23)、RASQNVNNWLN(SEQ NO.25)、RASQNINSWLN(SEQ NO.26)、RASQNINNWLN(SEQ NO.27)或RASQDVDSWLT(SEQ NO.29)中的任一种氨基酸序列；所述重链可变区HCDR2序列选自WVSFYQGNT(SEQ NO.59)、WVSFYNGQT(SEQ NO.60)或WVSFYNGNS(SEQ NO.61)中的任一种氨基酸序列；所述轻链可变区LCDR2序列选自GASTLES(SEQ NO.34)、AASTRET(SEQ NO.35)、GASSRQS(SEQ NO.36)、GASTRPT(SEQ NO.37)或GASNLAS(SEQ NO.39)中的任一种氨基酸序列；所述重链可变区HCDR3序列选自ARGYSLDV(SEQ NO.63)、ARGYGMSI(SEQ NO.64)、 ARGFGMDR(SEQ NO.65)或ARGYGMTV(SEQ NO.66)中的任一种氨基酸序列；所述轻链可变区LCDR3序列选自QQDNDIPLT(SEQ NO.43)、QQDNDMPLT(SEQ NO.45)、QQWFDVPTT(SEQ NO.46)、QQWDDTPNT(SEQ NO.47)或QQDSKIPLT(SEQ NO.49)中的任一种氨基酸序列。
其中，本发明还提供了多种包含上述轻链或上述重链的抗体、多肽或蛋白。
其中，本发明还提供了多种包含上述轻链或上述重链的抗体，所述抗体能够特异性与PCSK9结合，阻断PCSK9与LDLR的结合，提高细胞表面LDLR的数量或血液循环系统中LDLR的水平，降低血液循环系统中LDL或LDL-C的水平。
其中，本发明还提供了多种包含上述轻链或上述重链的多核苷酸序列或组合。
其中，所述抗PCSK9的单克隆抗体的重链恒定区包含IgG1、IgG2、IgG3和IgG4；所述轻链恒定区包含Cκ或C λ。
其中，所述重链恒定区优选IgG4或IgG2；所述重链的真核表达载体或原核生物表达载体。
其中，所述轻链恒定区优选Cκ；所述轻链的真核表达载体或原核生物表达
载体。
其中，本发明还提供了一种包含上述多核苷酸序列或组合的重组DNA表达载体；所述重组DNA表达载体的DNA序列中包含编码抗PCSK9抗体的上述重链可变区、重链恒定区、轻链可变区和轻链恒定区的氨基酸序列。
其中，本发明还提供了一种转染上述重组DNA表达载体的宿主细胞，所述宿主细胞包含原核细胞、酵母、昆虫或哺乳动物细胞。
其中，所述原核细胞优选大肠杆菌。
其中，所述哺乳动物细胞优选HEK293E细胞、CHO细胞或NS0细胞。
其中，本发明还提供了多种含有上述轻链或上述重链的全长抗体、单链抗体、单域抗体、双特异抗体、抗体药物偶联物。
其中，本发明还提供了多种含有上述轻链或上述重链的单克隆抗体、人工载体、药物或药物组合物。
其中，所述单克隆抗体包含全长抗体和抗PCSK9单克隆抗体的片段，所述片 段包含但不限于Fab、Fab’、F(ab’) 2、Fv或ScFv。
其中，本发明还提供了一种含有上述轻链或上述重链的检测试剂或试剂盒。
本发明所述抗体可用于通过消除、抑制或降低PCSK9活性而被改善、缓解、抑制或预防的疾病；所述疾病包含血脂异常、心脑血管疾病和血栓闭塞性疾病。
其中，所述血脂异常包含胆固醇升高、甘油三脂升高、低密度脂蛋白升高和高密度脂蛋白降低。所述心血管疾病包含冠状动脉硬化性心脏病、急性心肌梗塞、动脉粥状硬化、中风和外周动脉闭塞性疾病。
本发明一种抗PCSK9的单克隆抗体的获得方法，包括如下步骤：
(1)、抗PCSK9单链抗体的生物淘选，通过三轮抗体库的富集筛选，从全合成的ScFv噬菌体库中获得了一种亲和力较高的抗体序列DFSK9-1，其重链DFSK9-H1具有SEQ NO.1的氨基酸序列，其轻链DFSK9-L1具有SEQ NO.11的氨基酸序列；
(2)、以DFSK9-1为基础，通过计算机三级结构模拟，设计并构建轻链互补决定区CDR1、CDR2、CDR3突变抗体库,并对这个突变抗体库进行生物淘选和阳性克隆的筛选及鉴定，获得了10种含有不同轻链的单链抗体序列，分别命名为DFSK9-2、DFSK9-3、DFSK9-4、DFSK9-5、DFSK9-6、DFSK9-7、DFSK9-8、DFSK9-9、DFSK9-10、DFSK9-11，对应的轻链可变区分别命名为DFSK9-L2、DFSK9-L3、DFSK9-L4、DFSK9-L5、DFSK9-L6、DFSK9-L7、DFSK9-L8、DFSK9-L9、DFSK9-L10和DFSK9-L11，其对应的氨基酸序列分别见SEQ NO.12、SEQ NO.13、SEQ NO.14、SEQ NO.15、SEQ NO.16、SEQ NO.17、SEQ NO.18、SEQ NO.19、SEQ NO.20和SEQ NO.21；将上述单链抗体在噬菌体水平上进行亲和力比较；
(3)、选出5株亲和力较高的克隆DFSK9-2、DFSK9-4、DFSK9-5、DFSK9-6和DFSK9-8，设计并构建重链互补决定区CDR1、CDR2、CDR3突变抗体库，再对这个重链突变的抗体库进行生物淘选和阳性克隆的筛选及鉴定，获得了10种不同的单链抗体序列，分别命名为DFSK9-12、DFSK9-13、DFSK9-14、DFSK9-15、DFSK9-16、DFSK9-17、DFSK9-18、DFSK9-19、DFSK9-20和DFSK9-21；其中，DFSK9-12、DFSK9-13、DFSK9-14、DFSK9-15、DFSK9-16、DFSK9-17、DFSK9-18、DFSK9-19、DFSK9-20和DFSK9-21的轻链可变区序列分别是DFSK9-L2、DFSK9-L8、 DFSK9-L5、DFSK9-L6、DFSK9-L5、DFSK9-L6、DFSK9-L5、DFSK9-L4、DFSK9-L6、DFSK9-L6；其对应的氨基酸序列分别见SEQ NO.12、SEQ NO.18、SEQ NO.15、SEQ NO.16、SEQ NO.15、SEQ NO.16、SEQ NO.15、SEQ NO.14、SEQ NO.16、SEQ NO.16；DFSK9-12、DFSK9-13、DFSK9-14、DFSK9-15、DFSK9-16、DFSK9-17、DFSK9-18、DFSK9-19、DFSK9-20和DFSK9-21的重链可变区序列分别是DFSK9-H2、DFSK9-H8、DFSK9-H7、DFSK9-H3、DFSK9-H6、DFSK9-H4、DFSK9-H4、DFSK9-H9、DFSK9-H5、DFSK9-H10；其对应的氨基酸序列分别见SEQ NO.2、SEQ NO.8、SEQ NO.7、SEQ NO.3、SEQ NO.6、SEQ NO.4、SEQ NO.4、SEQ NO.9、SEQ NO.5、SEQ NO.10；将上述单链抗体在噬菌体水平上进行亲和力比较；
(4)、将(3)中所述单克隆重链可变区基因和轻链可变基因克隆到真核表达载体，转染宿主细胞，获得抗PCSK9单克隆抗体的全抗。
其中，所述CDR为互补决定区(complementarity-determining region)；所述ScFv为单链抗体(single-chain fragment variable)；所述ADCs为抗体药物偶联物(antibody-drug conjugates)；所述LDLR为低密度脂蛋白受体(Low Density Lipoprotein Receptor)；所述LDL-C为低密度脂蛋白胆固醇(Low Density Lipoprotein-Cholesterol)；所述HEK293E细胞为人胚肾293E细胞(human embryonic kidney 293E cell)；CHO细胞为中国仓鼠卵巢细胞(chinese hamster ovary cell)；NS0细胞为小鼠NS0胸腺瘤细胞。
本发明相对于现有技术而言，具备的有益效果是：
本发明提供的是多种全新的抗PCSK9抗体，与底物结合的亲和力高，能够很好的阻断PCSK9与LDLR的结合，并且影响细胞表面的LDLR的分布和表达，从而提高细胞表面的LDL-R与LDL结合能力，增加细胞对LDL摄取和降解，达到降低胞外的LDL和LDL-C的含量，达到降低LDL、LDL-C和总胆固醇目的。
本发明提供的单克隆抗体可以用于通过消除、抑制或降低PCSK9活性而改善、缓解、抑制或预防的疾病；所述疾病包含血脂异常、心脑血管疾病和血栓闭塞性疾病；血脂异常包含胆固醇升高、甘油三脂升高、低密度脂蛋白升高和高密度脂蛋白降低等；心血管疾病包含冠状动脉硬化性心脏病、急性心肌梗塞、动脉粥状硬化、中风和外周动脉闭塞性疾病等。
说明书附图
图1、pScFvDisb-S1质粒图谱；
图2、轻链突变抗体库阳性克隆噬菌体单克隆ELISA鉴定单链抗体的相对亲和力；
图3、轻链突变抗体库阳性克隆噬菌体梯度ELISA比较单链抗体的相对亲和力；
图4、重链突变抗体库阳性克隆噬菌体单克隆ELISA鉴定单链抗体的相对亲和力；
图5、重链突变抗体库阳性克隆噬菌体梯度ELISA比较单链抗体的相对亲和力；
图6、pTSEG4质粒图谱；
图7、pTSEK质粒图谱
图8、抗PCSK9全抗体与PCSK9在分子水平上结合试验；
图9、抗PCSK9全抗体竞争抑制LDLR与PCSK9结合实验；
图10、抗PCSK9全抗体生物学活性实验。
具体实施方式
本发明详细的实施方法参见实施例，实施例中所述的实验方法和试剂，若无特殊说明均为常规实验方法和试剂。以下实施例仅用于说明和解释本发明，而不是以任何方式限制本发明。
本发明提供了一种特异性结合PCSK9的单克隆抗体，所述重链可变区序列包含SEQ NO.1、2、3、4、5、6、7、8、9和10中的任一种,所述轻链可变区序列包含SEQ NO.11、12、13、14、15、16、17、18、19、20和21中的任一种。
优选地，所述特异性结合PCSK9的单克隆抗体的重链可变区序列选自SEQ NO.2、3、4、5、6、7、8、9和10中的任一种，所述轻链可变区序列选自SEQ NO.12、14、15、16和18中的任一种。
通过轻链噬菌体库筛选，所述抗体轻链或其功能性片段的轻链互补决定区LCDR1、LCDR2和LCDR3的氨基酸序列选自以下氨基酸序列中的任一组合(如表1所示)：
表1、轻链各个CDR区的氨基酸序列
Figure PCTCN2017119822-appb-000001
Figure PCTCN2017119822-appb-000002
通过重链噬菌体库筛选，所述抗体重链或其功能性片段重链的互补决定区CDR1、CDR2和CDR3分别用HCDR1、HCDR2和HCDR3表示：HCDR1为GGTFTNNA(SEQ NO.53)、GYTVTSYG(SEQ NO.54)或GYSLTSYG(SEQ NO.55)中的任一种；HCDR2为RIIPMFGMA(SEQ NO.56)、WLSFYNGNT(SEQ NO.57)、WVTFYNGNT(SEQ NO.58)、WVSFYQGNT(SEQ NO.59)、WVSFYNGQT(SEQ NO.60)或WVSFYNGNS(SEQ NO.61)中的任一种；HCDR3为AREGIPMI(SEQ NO.62)、ARGYSLDV(SEQ NO.63)、ARGYGMSI(SEQ NO.64)、ARGFGMDR(SEQ NO.65)、ARGYGMTV(SEQ NO.66)或ARGFGLSV(SEQ NO.67)中的任一种。
优选地，通过重链噬菌体库筛选，所述特异性结合PCSK9的单克隆抗体包含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；其中,所述重链可变区HCDR1序列为选自GYTVTSYG(SEQ NO.54)或GYSLTSYG(SEQ NO.55)的氨基酸序列；所述轻链可变区LCDR1序列选自RASQSVRNWLD(SEQ NO.23)、RASQNVNNWLN(SEQ NO.25)、RASQNINSWLN(SEQ NO.26)、RASQNINNWLN(SEQ NO.27)或RASQDVDSWLT(SEQ NO.29)中的任一种氨基酸序列；所述重链可变区HCDR2序列选自WVSFYQGNT(SEQ NO.59)、WVSFYNGQT(SEQ NO.60)或WVSFYNGNS(SEQ NO.61)中的任一种氨基酸序列；所述轻链可变区LCDR2序列选自GASTLES(SEQ NO.34)、AASTRET(SEQ NO.35)、GASSRQS(SEQ NO.36)、GASTRPT(SEQ NO.37)或GASNLAS(SEQ NO.39)中的一种氨基酸序列；所述重链可变区HCDR3序列选自ARGYSLDV(SEQ NO.63)、ARGYGMSI(SEQ NO.64)、ARGFGMDR(SEQ NO.65)或ARGYGMTV(SEQ NO.66)中的任一种氨基酸序列；所述轻链可变区LCDR3序列选自QQDNDIPLT(SEQ NO.43)、QQDNDMPLT(SEQ NO.45)、QQWFDVPTT(SEQ NO.46)、QQWDDTPNT(SEQ NO.47)或QQDSKIPLT(SEQ NO.49)中的任一种氨基酸序列。
本发明用全合成ScFv单链噬菌抗体库获得特异性抗体的方法，所述全人源的特异性结合PCSK9的单克隆抗体是用噬菌体抗体库技术筛选而得，其步骤在于：
(1)、抗PCSK9单链抗体的生物淘选，通过三轮抗体库的富集筛选，获得了一种亲和力较高的抗体序列DFSK9-1；
(2)、以DFSK9-1为基础，通过计算机辅助设计，构建轻链CDR123突变库并对该抗体库进行生物淘选和阳性克隆的筛选及鉴定，获得了10种不同轻链的抗体序列的克隆DFSK9-2、DFSK9-3、DFSK9-4、DFSK9-5、DFSK9-6、DFSK9-7、DFSK9-8、DFSK9-9、DFSK9-10、DFSK9-11。将上述10种单链抗体在噬菌体水平进行亲和力比较；
(3)、选出两株亲和力较高的克隆，构建重链CDR123库并对该库进行生物淘选和阳性克隆的筛选，获得了种10不同序列的单链抗体DFSK9-12、DFSK9-13、DFSK9-14、DFSK9-15、DFSK9-16、DFSK9-17、DFSK9-18、DFSK9-19、DFSK9-20、DFSK9-21；将上述单链抗体在噬菌体水平上进行亲和力比较；
(4)、将(3)中所述单克隆重链可变区基因和轻链可变基因克隆到真核表达载体，转染宿主细胞，获得抗PCSK9单克隆抗体的全抗。
优选地，对(4)的抗PCSK9单克隆抗体的全抗再进行亲和力和生物活性试验。
具体实施例
以下结合附图和实施例详述本发明。
实施例1、抗PCSK9单链抗体的生物淘选
采用基因克隆的方法改造pCom3载体，改造后的载体命名为pScFvDisb-S1(图1)。以该载体为基础构建全合成噬菌体抗体库。
免疫管中包被抗原PCSK9-His 10μg/1ml/管，4℃包被过夜。用PBST-4％milk分别封闭免疫管和噬菌体抗体库(噬菌体投入量约为10 9-10 12)，37℃封闭1h。封闭后的噬菌体抗体库加入免疫管中进行抗原抗体结合，37℃反应1h。PBST-PBS洗去未结合的噬菌体，0.1M pH2.2的Glycine-HCl洗脱，用1.5M pH8.8的Tris-HCl中和洗脱液至pH7.0左右。洗脱液感染10ml生长至OD值约0.5-0.8的XL1-Blue菌液，37℃先静置30min后150rpm摇床振荡培养1h。取出1％菌液进行梯度稀释，涂布于2YTATG小平皿上，用于计算噬菌体产出量。剩余的菌液离心后涂布于2YTATG大平皿，37℃过夜培养。将过夜培养菌转接至2YTATG液体 培养基，摇至对数期后加入M13K07辅助噬菌体感染，28℃培养过夜扩增噬菌体，PEG6000-NaCl沉降纯化噬菌体用于下一轮筛选。共进行3轮噬菌体库富集筛选。
实施例2、抗PCSK9单链抗体阳性克隆的筛选
经过三轮筛选后，挑取分隔良好的单克隆菌落，接种于加有2YTATG液体培养基的96孔深孔板，37℃，220rpm培养约5h至其对数生长期，每孔加入约10 10的辅助噬菌体M13KO7，37℃静置30min后150rpm振荡培养1h。4000rpm，离心15min，沉淀重悬于2YTATKA液体培养基中，28℃，220rpm培养过夜。4000rpm，4℃离心15min，吸取含噬菌体上清进行单克隆ELISA鉴定。筛选得到亲和力较高的单链抗体DFSK9-1，其重链可变区命名为DFSK9-H1,其氨基酸序列见SEQ NO.1；其轻链可变区命名为DFSK9-L1,氨基酸序列见SEQ NO.11；
SEQ NO.1(DFSK9-H1重链可变区序列)：
Figure PCTCN2017119822-appb-000003
SEQ NO.11(DFSK9-L1轻链可变区序列)：
Figure PCTCN2017119822-appb-000004
实施例3、对单链抗体DFSK9-1进行体外亲和力成熟
3.1.构建DFSK9-1轻链CDR123突变库
用NheI和NotI对pScFvDisb-DFSK9-1质粒进行双酶切，酶切产物琼脂糖凝胶电泳后，切胶回收大小5.5kb的条带；用NheI和NotI对合成的轻链突变库基因VLCDR123M进行双酶切，通用产物回收试剂盒回收产物。突变库基因与载体按摩尔比3：1比例经T4DNA连接酶16℃连接4h。将连接产物电击法转化至XL1-Blue电转感受态中。37℃，150rpm振动培养1h复苏。取1％菌液，稀释后涂小平皿，计算库容量。其余菌液4000rpm离心15min后，沉淀涂布于2YTATG大平皿上，37℃倒置培养过夜。建成的抗体库库容大约为10 8，随机挑取20个克隆进行序列分析，序列正确率和多样性都大于90％。
3.2.噬菌体抗体库的生物淘选和阳性克隆的筛选
在按照实施例1和实施例2的方法进行生物淘选和阳性克隆筛选，对亲和力较高的克隆进行测序，共得到10种不同的单链抗体序列，分别命名为DFSK9-2、DFSK9-3、DFSK9-4、DFSK9-5、DFSK9-6、DFSK9-7、DFSK9-8、DFSK9-9、DFSK9-10、 DFSK9-11，对应的轻链可变区命名为DFSK9-L2、DFSK9-L3、DFSK9-L4、DFSK9-L5、DFSK9-L6、DFSK9-L7、DFSK9-L8、DFSK9-L9、DFSK9-L10、DFSK9-L11，它们对应的氨基酸序列分别见SEQ NO.12、SEQ NO.13、SEQ NO.14、SEQ NO.15、SEQ NO.16、SEQ NO.17、SEQ NO.18、SEQ NO.19、SEQ NO.20和SEQ NO.21；轻链可变区序列SEQ NO.12-SEQ NO.21如下：
SEQ NO.12(DFSK9-L2轻链可变区序列)：
Figure PCTCN2017119822-appb-000005
SEQ NO.13(DFSK9-L3轻链可变区序列)：
Figure PCTCN2017119822-appb-000006
SEQ NO.14(DFSK9-L4轻链可变区序列)：
Figure PCTCN2017119822-appb-000007
SEQ NO.15(DFSK9-L5轻链可变区序列)：
Figure PCTCN2017119822-appb-000008
SEQ NO.16(DFSK9-L6轻链可变区序列)：
Figure PCTCN2017119822-appb-000009
SEQ NO.17(DFSK9-L7轻链可变区序列)：
Figure PCTCN2017119822-appb-000010
SEQ NO.18(DFSK9-L8轻链可变区序列)：
Figure PCTCN2017119822-appb-000011
SEQ NO.19(DFSK9-L9轻链可变区序列)：
Figure PCTCN2017119822-appb-000012
SEQ NO.20(DFSK9-L10轻链可变区序列)：
Figure PCTCN2017119822-appb-000013
Figure PCTCN2017119822-appb-000014
SEQ NO.21(DFSK9-L11轻链可变区序列)：
Figure PCTCN2017119822-appb-000015
单克隆噬菌体ELISA鉴定结果如图2所示；
3.3.噬菌体水平梯度稀释ELISA鉴定抗PCSK9单链抗体的相对亲和力
将本实施例3.2中获得的克隆进行单克隆噬菌体的展示和纯化，进行噬菌体水平的梯度稀释ELISA鉴定单链抗体的相对亲和力。
用pH7.2的0.01M PBS缓冲液包被PCSK9-His(300ng/孔/100μl)，4℃包被过夜。PBST洗涤3次，PBST-4％milk 37℃封闭1h。加入PBST-4％milk五倍梯度稀释的纯化噬菌体样品(100μl/孔)，37℃静置1h。用PBST洗涤5次，加入PBST-4％milk 1:5000稀释的anti-M13-HRP单克隆抗体(100μl/孔)，37℃放置1h。TMB显色试剂盒显色(100μl/孔)，室温显色10min，用2M H 2SO 4(50μl/孔)终止显色,。酶标仪波长450nm和630nm读数。使用软件GraphPad Prism 5Demo分析数据并作图，结果如图3所示：结果显示筛选出的噬菌体单链抗体均能与PCSK9进行结合，而且DFSK9-2、DFSK9-4、DFSK9-5、DFSK9-6和DFSK9-8的亲和力明显高于其它克隆，选取上述5个单链抗体进行下一步试验。
实施例4、对筛选出的抗PCSK9的单链抗体再次进行体外亲和力成熟
4.1链置换法构建重链CDR123突变库
用NcoI-HF和KpnI对实施例3.3中DFSK9-2、DFSK9-4、DFSK9-5、DFSK9-6和DFSK9-8等5种单链抗体的混合质粒进行双酶切，切胶回收大小5.5kb的条带；用NcoI-HF和KpnI对合成的重链突变库基因VHCDR123M进行双酶切，通用回收试剂盒回收酶切产物。突变库基因与载体按摩尔比3：1比例经T4DNA连接酶16℃连接4h。将连接产物电击法转化至XL1电转感受态中。37℃，150rpm培养1h复苏。取1％菌液，稀释后涂小平皿，计算库容量。其余菌液4000rpm，离心15min后，沉淀涂布于2YTATG大平皿上，37℃倒置培养过夜。构建成的抗体库库容大约为5*10 8，随机挑取20个克隆进行序列分析，正确率和多样性均大于90％。
4.2噬菌体抗体库的生物淘选和阳性克隆的筛选
将实施例4.1中的抗体库进行噬菌体展示和纯化回收。从该库中淘选抗PCSK9的单链抗体。噬菌体抗体库的生物淘选方法和阳性克隆的筛选同实施案例 1和实施例2。测序后发现共筛选到10种不同的抗PCSK9抗体序列，分别命名为DFSK9-12、DFSK9-13、DFSK9-14、DFSK9-15、DFSK9-16、DFSK9-17、DFSK9-18、DFSK9-19、DFSK9-20、DFSK9-21。其中DFSK9-12的轻链可变区序列是DFSK9-L2，对应的氨基酸序列见SEQ NO.12；DFSK9-13的轻链可变区序列是DFSK9-L8，对应的氨基酸序列见SEQ NO.18；DFSK9-14、DFSK9-16和DFSK9-18的轻链可变区序列是DFSK9-L5，对应的氨基酸序列见SEQ NO.15；DFSK9-15、DFSK9-17、DFSK9-20和DFSK9-21的轻链可变区序列是DFSK9-L6，对应的氨基酸序列见SEQ NO.16；DFSK9-19的轻链可变区序列是DFSK9-L4，对应的氨基酸序列见SEQ NO.14。DFSK9-12的重链可变区序列是DFSK9-H2，对应的氨基酸序列见SEQ NO.2；DFSK9-13的重链可变区序列是DFSK9-H8，对应的氨基酸序列见SEQ NO.8；DFSK9-14的重链可变区序列是DFSK9-H7，对应的氨基酸序列见SEQ NO.7；DFSK9-15的重链可变区序列是DFSK9-H3，对应的氨基酸序列见SEQ NO.3；DFSK9-16的重链可变区序列是DFSK9-H6，对应的氨基酸序列见SEQ NO.6；DFSK9-17和DFSK9-18的重链可变区序列是DFSK9-H4，，对应的氨基酸序列见SEQ NO.4；DFSK9-19的重链可变区是DFSK9-H9，对应的氨基酸序列见SEQ NO.9；DFSK9-20的重链可变区是DFSK9-H5，对应的氨基酸序列见SEQ NO.5；DFSK9-21的重链可变区序列是DFSK9-H10，对应的氨基酸序列见SEQ NO.10。轻链可变区序列参照实施例3.2中氨基酸序列SEQ NO.12、SEQ NO.14、SEQ NO.15、SEQ NO.16和SEQ NO.18，重链可变区氨基酸序列SEQ NO.2-SEQ NO.10如下：
SEQ NO.2(DFSK9-H2重链可变区序列)：
Figure PCTCN2017119822-appb-000016
SEQ NO.3(DFSK9-H3重链可变区序列)：
Figure PCTCN2017119822-appb-000017
SEQ NO.4(DFSK9-H4重链可变区序列)：
Figure PCTCN2017119822-appb-000018
SEQ NO.5(DFSK9-H5重链可变区序列)：
Figure PCTCN2017119822-appb-000019
SEQ NO.6(DFSK9-H6重链可变区序列)：
Figure PCTCN2017119822-appb-000020
SEQ NO.7(DFSK9-H7重链可变区序列)：
Figure PCTCN2017119822-appb-000021
SEQ NO.8(DFSK9-H8重链可变区序列)：
Figure PCTCN2017119822-appb-000022
SEQ NO.9(DFSK9-H9重链可变区序列)：
Figure PCTCN2017119822-appb-000023
SEQ NO.10(DFSK9-H10重链可变区序列)：
Figure PCTCN2017119822-appb-000024
单克隆phage ELISA鉴定phage-Abs的相对亲和力如图4所示。
4.3.梯度稀释噬菌体ELISA鉴定抗PCSK9单链抗体的亲和力
将本实施例4.2中获得的克隆进行单克隆噬菌体的展示和纯化，进行噬菌体梯度稀释ELISA实验鉴定单链抗体的亲和力，方法同实施例3中3.3。结果如图5所示：筛选出的10种不同的单链抗体均能与PCSK9进行很好的结合，且亲和力均高于最初的单链DFSK9-1。
实施例5、抗PCSK9全抗体的亲和力鉴定
5.1抗PCSK9全抗体的制备
将实施例4中筛到抗体的重链VH克隆至含有重链恒定区基因(γ4)的载体pTSEG4(图6)上，轻链VK基因克隆到含有轻链恒定区基因(κ链)的载体pTSEK(图7)上，载体pTSEG4和pTSEK均以PTT载体为基础改造获得。PTT载体的制备过程在参考文献(Yves.Durocher,Sylvie.Perret and Amine.Kamen Nucleic Acids Research，2002Vol.30，No.2e9)中有详细描述。瞬时转染HEK293E细胞，进行全抗体表达。使用AKTA仪器protein A亲和柱纯化获得全抗体蛋白。
5.2全抗体与PCSK9的结合实验
用pH7.2的0.01M PBS缓冲液包被PCSK9-His(300ng/孔/100μl)，4℃包被过夜。用300μl/孔PBST(1‰Tween 20)洗3次，再加入PBST-4％milk 37℃封闭1h。加入不同稀释度的全抗体DFSK9-1，DFSK9-12、DFSK9-13、DFSK9-14、DFSK9-15、DFSK9-16、DFSK9-17、DFSK9-18、DFSK9-19、DFSK9-20和DFSK9-21。11种全抗体最高浓度是50ug/ml，5倍梯度稀释，每个全抗体做8个梯度，37℃孵育1h。用300μl/孔PBST洗5次，再加入用PBST-4％milk 1:5000稀释的山羊抗人IgG-HRP二抗37℃孵育1h。用300μl/孔PBST洗5次，TMB显色试剂盒显色(100μl/孔)，室温显色10min，然后用2M H 2SO 4(50μl/孔)终止显色。酶标仪波长450nm和630nm读数。使用软件GraphPad Prism 5Demo分析数据作图，实验结果如图8和表2所示，所有抗体均能很好的与PCSK9分子结合,其中DFSK9-14、DFSK9-15、DFSK9-16、DFSK9-17、DFSK9-18、DFSK9-20和DFSK9-21具有相对较高的亲和力。
表2、全抗体亲和力EC 50值
Figure PCTCN2017119822-appb-000025
5.3全抗体竞争抑制LDLR与PCSK9结合实验
用pH7.2的0.01M PBS包被LDLR-Fc(100ng/孔/100μl)，4℃包被过夜。PBST 洗涤3次，再加入PBST-4％milk 37℃封闭1h。加入PBST-4％milk稀释的浓度为2μg/ml的PCSK9-His(100μl/孔)，37℃孵育1h。加入PBST-4％milk稀释不同稀释度的全抗体DFSK9-1，DFSK9-12、DFSK9-13、DFSK9-14、DFSK9-15、DFSK9-16、DFSK9-17、DFSK9-18、DFSK9-19、DFSK9-20和DFSK9-21。11种全抗体最高浓度是100ug/ml，五倍梯度稀释，每个全抗体做8个梯度，37℃孵育2h。PBST洗5次，加入用PBST-4％milk稀释的小鼠抗His IgG-HRP二抗，37℃孵育1h。TMB显色试剂盒显色(100μl/孔)，室温显色10min，用2M H 2SO 4(50μl/孔)终止显色。酶标仪波长450nm和630nm读数。使用软件GraphPad Prism 5 Demo作图，实验结果如图9和表3所示，所有抗体均能有效的抑制PCSK9与LDLR的结合，其中DFSK9-14、DFSK9-15、DFSK9-16、DFSK9-17、DFSK9-18、DFSK9-20和DFSK9-21抑制能力稍强一些。
表3、全抗体竞争实验IC 50值
Figure PCTCN2017119822-appb-000026
5.4BIAcore X100测定全抗体的亲和力
采用捕获法测定全抗体的亲和力。将山羊抗人IgG偶联到CM5芯片表面，分别稀释DFSK9-1、DFSK9-12、DFSK9-13、DFSK9-14、DFSK9-15、DFSK9-16、DFSK9-17、DFSK9-18、DFSK9-19、DFSK9-20和DFSK9-21保证大约200RU左右的抗体被山羊抗人IgG捕获。将PCSK9设置一系列的浓度梯度(200nM、100nM、50nM、25nM、12.5nM、6.25nM、3.125nM、1.5625nM、0.78125nM)流经固定相表面，测定抗体的亲和力。结果发现筛出的抗体都具有较高的亲和力(见表4)，选取亲和力最高的8种全抗体进行生物学活性实验。
表4、抗PCSK9全抗体亲和力常数测定数值
No. Sample ka(1/Ms) kd(1/s) KD(M)
1 DFSK9-1 5.178E+5 9.191E-5 1.755E-10
2 DFSK9-12 4.180E+5 4.975E-5 1.190E-10
3 DFSK9-13 5.747E+5 2.990E-5 5.202E-11
4 DFSK9-14 2.700E+6 7.901E-5 2.926E-11
5 DFSK9-15 5.425E+5 4.746E-6 8.749E-12
6 DFSK9-16 1.217E+6 5.613E-5 4.613E-11
7 DFSK9-17 1.184E+6 1.844E-6 1.557E-12
8 DFSK9-18 5.117E+5 1.992E-6 3.893E-12
9 DFSK9-19 3.952E+5 7.339E-5 1.857E-10
10 DFSK9-20 9.252E+5 1.451E-5 1.568E-11
11 DFSK9-21 5.918E+5 4.239E-6 7.162E-12
实例6、抗PCSK9全抗体生物学活性实验
将HepG2细胞以2.5X10 5cells/ml接种到96孔中。次日，将10％FBS的EMEM生长培养基换成80μl含5％除脂蛋白FBS的分析培养基，37℃培养24h。次日，将分析培养基稀释的不同稀释度的全抗体(10μl/孔)加入到已接种HepG2细胞的培养板中。8种全抗体样品初始浓度为900nmol/L，3倍梯度稀释，每个全抗体做8个梯度。然后再加入30nmol/L的PCSK9溶液(10μl/孔)，混匀后37℃，5％CO2条件下培养4h。每孔加入10ul 0.1mg/m L BODIPY标记的LDL溶液，混匀后37℃，5％CO2条件下继续培养15-20h。洗涤每孔加入200μl PBS洗涤2次。每孔加入100μl PBS，酶标仪波长490nm和520nm读取相对荧光单位RFU数值。GraphPad Prism 5Demo软件分析数据作图，结果如图10所示，以同型IgG为阴性对照，由图上可以看出8个抗体均能以剂量依赖性方式阻断PCSK9与低密度脂蛋白受体LDLR结合，增加低密度脂蛋白LDL在HepG2细胞内摄取率，各抗体之间的生物学活性相差不多。
表5、生物学活性实验EC 50值
No. Sample EC 50(nmol/L) No. Sample EC 50(nmol/L)
1 DFSK9-13 29.61 7 DFSK9-17 18.51
2 DFSK9-14 16.59 8 DFSK9-18 23.91
3 DFSK9-15 15.31 9 DFSK9-20 17.95
4 DFSK9-16 23.80 10 DFSK9-21 20.33
对于本领域的普通技术人员而言，具体实施例只是对本发明进行了示例性描述，显然本发明具体实现并不受上述方式的限制，只要采用了本发明的方法构思和技术方案进行的各种非实质性的改进，或未经改进将本发明的构思和技术方案直接应用于其它场合的，均在本发明的保护范围之内。

Claims (17)

  1. 一种抗PCSK9的单克隆抗体，其特征在于，包括：轻链和重链；所述轻链的互补决定区CDR1、CDR2和CDR3分别用LCDR1、LCDR2和LCDR3表示；并且所述重链的互补决定区CDR1，CDR2和CDR3分别用HCDR1、HCDR2和HCDR3表示；LCDR1包含RASQSIDNRLT、RASQSVRNWLD、RASQGINSWLN、RASQNVNNWLN、RASQNINSWLN、RASQNINNWLN、RASQGIHNWLN、RASQDVDSWLT、RASQSVRNWLN、RASQDVRNWLT或RASQSIRSYLN中的任一种；LCDR2包含DASSRQS、GASTLES、AASTRET、GASSRQS、GASTRPT、DASNRQS、GASNLAS、DASNLQS或DASSRPT中的任一种；LCDR3包含QQPENDPTT、QQDNDIPLT、QQWNNTPNT、QQDNDMPLT、QQWFDVPTT、QQWDDTPNT、QQNSNIPLT、QQDSKIPLT、QQWTDTPLT、QQDDSTPPT或QQGDSMPMT中的任一种；HCDR1包含GGTFTNNA、GYTVTSYG或GYSLTSYG中的任一种；HCDR2包含RIIPMFGMA、LSFYNGNT、VTFYNGNT、VSFYQGNT、VSFYNGQT或VSFYNGNS中的任一种；HCDR3包含AREGIPMI、ARGYSLDV、ARGYGMSI、ARGFGMDR、ARGYGMTV或ARGFGLSV中的任一种。
  2. 根据权利要求1所述的抗PCSK9的单克隆抗体，其特征在于，所述轻链可变区氨基酸序列优选SEQ NO.11、SEQ NO.12、SEQ NO.13、SEQ NO.14、SEQ NO.15、SEQ NO.16、SEQ NO.17、SEQ NO.18、SEQ NO.19、SEQ NO.20或SEQ NO.21中的任一种。
  3. 根据权利要求1所述的抗PCSK9的单克隆抗体，其特征在于，所述重链可变区氨基酸序列优选SEQ NO.1、SEQ NO.2、SEQ NO.3、SEQ NO.4、SEQ NO.5、SEQ NO.6、SEQ NO.7、SEQ NO.8、SEQ NO.9或SEQ NO.10中的任一种。
  4. 根据权利要求1所述的抗PCSK9的单克隆抗体，其特征在于，所述重链可变区HCDR1序列选自GYTVTSYG或GYSLTSYG的氨基酸序列；所述轻链可变区LCDR1序列选自RASQSVRNWLD、RASQNVNNWLN、RASQNINSWLN、RASQNINNWLN或RASQDVDSWLT中的任一种氨基酸序列；所述重链可变区HCDR2序列选自VSFYQGNT、VSFYNGQT或VSFYNGNS中的任一种氨基酸序列；所述轻链可变区LCDR2序列选自GASTLES、AASTRET、GASSRQS、GASTRPT或GASNLAS中的任一种氨基酸序列；所述重链可变区HCDR3序列选自ARGYSLDV、ARGYGMSI、ARGFGMDR或ARGYGMTV中的任一种氨基酸序列；所述轻链可变区LCDR3序列选自QQDNDIPLT、QQDNDMPLT、QQWFDVPTT、QQWDDTPNT或QQDSKIPLT中的任一种氨基酸序列。
  5. 一种包含上述权利要求1-4任一项所述的轻链或重链的抗体、多肽或蛋白。
  6. 一种包含如权利要求1-4任一项所述的轻链或重链的氨基酸序列用于全长抗体、单链抗体、单域抗体、双特异抗体、抗体药物偶联物和嵌合抗原受体T细胞免疫疗法。
  7. 一种包含如权利要求1-4任一项所述的轻链或重链的抗体，所述抗体能够特异性与PCSK9结合。
  8. 一种编码如权利要求1-4任一项所述的重链或轻链的多核苷酸序列或组合。
  9. 一种包含如权利要求8所述的多核苷酸序列或组合的重组DNA表达载体。
  10. 一种转染如权利要求9所述的重组DNA表达载体的宿主细胞，所述宿主细胞包含原核细胞、酵母、昆虫或哺乳动物细胞。
  11. 根据权利要求10所述的宿主细胞，其特征在于，所述原核细胞优选大肠杆菌；所述哺乳动物细胞优选HEK293E细胞、CHO细胞或NS0细胞。
  12. 根据权利要求1-4任一项所述的抗PCSK9的单克隆抗体，其特征在于，所述抗PCSK9的单克隆抗体的重链恒定区包含IgG1、IgG2、IgG3或IgG4；所述轻链恒定区包含Cκ或C λ。
  13. 根据权利要求12所述的抗PCSK9的单克隆抗体，其特征在于，所述重链恒定区优选IgG4或IgG2；所述轻链恒定区优选Cκ。
  14. 一种包含如权利要求1-4任一项所述的轻链或重链的单克隆抗体、人工载体、药物或药物组合物。
  15. 根据权利要求14所述的单克隆抗体，其特征在于，所述单克隆抗体包含全长抗体或抗PCSK9单克隆抗体的片段，所述片段包含但不限于Fab、Fab’、F(ab’) 2、Fv和ScFv中的一种或几种的组合。
  16. 一种包含如权利要求1-4任一项所述的轻链或重链的检测试剂或试剂盒。
  17. 一种包含如权利要求1-4任一项所述的轻链或重链的抗体，用于消除、抑制或降低PCSK9活性及其改善、缓解、抑制或预防的疾病；所述疾病包含血脂异常、心脑血管疾病和血栓闭塞性疾病。
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CN109963877B (zh) * 2017-06-14 2023-01-20 江苏恒瑞医药股份有限公司 Pcsk9抗体、其抗原结合片段及其医药用途
CN109897110B (zh) * 2017-12-08 2022-05-17 深圳华大生命科学研究院 纳米抗体及其制备方法
CN110872353B (zh) * 2018-09-03 2021-05-04 深圳华大基因科技有限公司 特异性结合pcsk9抗原的纳米抗体及其制备方法和应用
CN110981962B (zh) * 2019-12-19 2022-07-12 中国药科大学 Pcsk9抗体、其抗原结合片段及其应用
CN111171152B (zh) * 2020-01-15 2023-04-18 吉林医药学院 Pcsk9抗体及其制备方法和应用
CN111620950B (zh) * 2020-06-16 2023-01-31 中国药科大学 全人源抗pcsk9抗体、其抗原结合片段及其应用
CN113150149B (zh) * 2020-06-19 2021-10-08 北京东方百泰生物科技股份有限公司 一种抗il-17ra单克隆抗体的纯化方法
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