CN111620950B

CN111620950B - Fully human anti-PCSK9 antibody, antigen binding fragment thereof and application thereof

Info

Publication number: CN111620950B
Application number: CN202010548397.3A
Authority: CN
Inventors: 谭树华; 徐梦龙; 汪可; 陈满满; 雷高新
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2020-06-16
Filing date: 2020-06-16
Publication date: 2023-01-31
Anticipated expiration: 2040-06-16
Also published as: CN111620950A

Abstract

The invention discloses a fully human anti-PCSK9 antibody or an antigen-binding fragment thereof and application thereof, and the anti-PCSK9 single-chain antibody is screened from a high-capacity fully human phage antibody library by taking human PCSK9 as an antigen. Then, a novel, high-activity and fully human anti-PCSK9 single-chain and full-length antibody is obtained by adopting in-vitro affinity maturation, CDR region cross cloning and antibody full-length technologies. The antibody and the fragment thereof can specifically bind to PCSK9, effectively inhibit PCSK 9-mediated degradation of a hepatocyte surface low-density lipoprotein receptor (LDLR), and remarkably enhance the uptake of low-density lipoprotein cholesterol (LDL-C) by hepatocytes, thereby remarkably reducing the serum LDL-C level. The antibody and the fragment thereof are applied to preparing medicaments for preventing and treating PCSK9 mediated diseases such as hypercholesterolemia, hyperlipidemia obesity, atherosclerosis, cardiovascular and cerebrovascular diseases and the like.

Description

Fully human anti-PCSK9 antibody, antigen binding fragment thereof and application thereof

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a novel fully human anti-Proprotein convertase subtilisin/kexin 9 (PCSK 9) antibody, an antigen binding fragment thereof and medical application thereof.

Background

Cardiovascular Disease (CVD) seriously compromises human health, while hypercholesterolemia, which is primarily characterized by elevated plasma low density lipoprotein cholesterol (LDL-C) levels, promotes atheromatous plaque formation and is an important risk factor for CVD induction (Nature reviews Disease polymers 2017, 3. The clearance of LDL-C in human blood is mainly completed by a liver cell surface Low Density Lipoprotein Receptor (LDLR), when the LDL-C is combined with the LDLR, an LDL-C/LDLR compound can be formed and enter lysosomes, and after the LDL-C is degraded, the LDLR can return to the surface of a cell membrane for recycling.

Proprotein convertase subtilisin/kexin 9 (pcsk 9) is a glycoprotein consisting of 692 amino acids, the ninth member of the Proprotein Convertase (PCs) family. PCSK9 is a secreted serine protease, mainly expressed in tissues such as liver and intestine, and then released into the blood (J Biol chem.2004;279 (47): 48865-48875; trends Biochem Sci.2007 (2): 71-77). PCSK9 specifically binds to the epidermal growth factor-like domain of LDLR on the surface of hepatocytes after entering the blood circulation, and directs it to reach lysosomes, where it is degraded, resulting in a decrease in LDLR on the surface of hepatocytes, thereby reducing the ability of the liver to clear LDL-C, and ultimately leading to increased levels of LDL-C in the blood and the development of hypercholesterolemia (Proc Natl Acad Sci U S A.2008;105 (35): 13045-13050 circulation research.2014 (6): 1022-1036. Therefore, drugs that inhibit PCSK9 synthesis, secretion and block its interaction with LDLR can promote LDL-C clearance and reduce the risk of hypercholesterolemia (Annu Rev Pharmacol Toxicol.2014; 54.

In addition, it has been reported in the literature that PCSK9 elevation is also closely related to obesity and type 2 Diabetes (Pediatr diabetes.2017.PMID:28093849, DOI.

The anti-PCSK9 antibody can specifically block the combination of PCSK9 and LDLR, inhibit PCSK 9-mediated degradation of LDLR, and increase the level of LDLR on the surface of liver cells, thereby enhancing the clearance function of the liver cells to LDL-C. Therefore, the development of anti-PCSK9 antibodies has attracted attention in recent years. The fully human monoclonal antibody Evolcumab developed by Amgen in the United states (AMG 145) and the fully human monoclonal antibody Alirocumab developed by Regeneron/Sanofi in the United states (REGN 727/SAR 236553) have been approved in the United states and Europe for treatment of heterozygote familial hypercholesterolemia (J Clin Pharmacol.2017;57 (1): 7-32) or treatment of cardiovascular high risk subjects where statins are not effective in reducing LDL-C.

The phage display technology (phase display) has the advantages of no need of antigen immunization, high screening efficiency and convenient affinity maturation, and is a main technical platform for discovering fully human antibodies at present. The invention takes recombinant human PCSK9 as antigen, anti-human PCSK9 single-chain antibody is screened from a fully human phage antibody library, and then a novel, high-activity and fully human anti-human PCSK9 antibody is obtained by in vitro affinity maturation and CDR region cross cloning (cross-cloning) technology (Molecular Immunology,2008,46 (1): 135-144). The antibody has the advantages of low immunogenicity, strong specificity and high activity, and can be used for diagnosing, preventing and treating PCSK 9-mediated hypercholesterolemia, hyperlipidemia, atherosclerosis, cardiovascular and cerebrovascular diseases, non-alcoholic fatty liver, obesity, nephropathy and other diseases.

Disclosure of Invention

The invention aims to: the invention aims to provide a novel and fully human anti-PCSK9 antibody and an antigen binding fragment thereof. The antibody and the antigen binding fragment thereof can specifically bind to PCSK9, and effectively inhibit degradation of PCSK9 on the surface LDLR of hepatocytes, thereby increasing the level of LDLR of hepatocytes and enhancing the absorption function of hepatocytes on LDL-C.

The invention also aims to provide application of the fully human anti-PCSK9 antibody and the antigen-binding fragment thereof in preparation of medicaments for preventing and treating diseases including but not limited to hypercholesterolemia, hyperlipidemia, atherosclerosis, cardiovascular and cerebrovascular diseases, fatty liver, obesity, nephropathy and the like.

The technical scheme is as follows: in order to achieve the purpose, the invention adopts the following technical scheme:

constructing a recombinant expression plasmid pTT5-hPCSK9 of human PCSK9, transiently transfecting CHO3E7 cells (Nanjing King Musry Biotechnology Co., ltd.) with the plasmid, carrying out suspension culture for 7 days, centrifugally collecting culture supernatant, and separating and purifying by adopting a nickel column affinity chromatography and a molecular exclusion chromatography method to obtain the recombinant human PCSK9 protein.

And (3) screening the anti-PCSK9 single-chain antibody from the high-capacity fully human phage antibody library by using the human PCSK9 protein as a target antigen. Degenerate oligonucleotide primers were designed by combining sequence alignment with KABAT database and mutation hotspot analysis, and semi-random mutations were introduced into six CDR regions, respectively, by whole plasmid PCR amplification. After the PCR amplification product is subjected to Dpn I enzyme digestion of a primary template, the Escherichia coli E.coli TG1 super competent cells are electrically transformed, a full-human anti-PCSK9 single-chain antibody mutant library is prepared, and 6 dominant mutants with high affinity and activity are obtained after phage display screening. The affinity and activity of the dominant mutants were further improved by using a CDR region cross-cloning method (Molecular immunology.2008; 46. On the basis, the obtained high-activity anti-PCSK9 single-chain antibody is subjected to full-length integration, and the in vitro and in vivo biological activity of the antibody is detected.

The antibody and the antigen binding fragment thereof can be specifically bound to PCSK9, effectively inhibit the interaction between PCSK9 and LDLR, block PCSK 9-mediated degradation of LDLR, and significantly enhance the uptake capacity of hepatocytes on LDL-C.

More specifically, the antibodies or antibody fragments of the invention comprise a heavy chain variable region and a light chain variable region, which in some embodiments are linked by a flexible peptide comprising 15 amino acids (GGGGSGGGGSGGGGS) to form a single chain antibody; in other embodiments, the heavy chain variable region and the light chain variable region are joined to a human IgG1 constant region to form a full-length antibody.

The complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain are respectively represented by HCDR1, HCDR2 and HCDR 3; the complementarity determining regions CDR1, CDR2 and CDR3 of the light chain are denoted by LCDR1, LCDR2 and LCDR3, respectively.

More specifically, HCDR1 comprises SEQ ID NO:1 to 6, or an amino acid sequence substantially identical to SEQ ID NO: 1-6, preferably at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity, or an amino acid sequence having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence;

HCDR2 comprises SEQ ID NO: 7. 8, or comprises an amino acid sequence substantially identical to any one of SEQ ID NOs: 7. 8, preferably at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity, or an amino acid sequence having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence;

HCDR3 comprises SEQ ID NO:9 to 12, or comprises an amino acid sequence identical to any one of SEQ ID NOs: 9-12, preferably at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity, or an amino acid sequence having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence;

LCDR1 comprises SEQ ID NO: 13-15, or comprises an amino acid sequence identical to any one of SEQ ID NOs: 13-15, preferably at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity, or an amino acid sequence having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence;

LCDR2 comprises SEQ ID NO:16 or comprises an amino acid sequence substantially identical to SEQ ID NO:16, preferably at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity, or an amino acid sequence having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence;

LCDR3 comprises SEQ ID NO:17 to 24, or a sequence substantially identical to any one of the amino acid sequences shown in SEQ ID NOs: 17-24, preferably at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity, or an amino acid sequence having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence.

The heavy chain variable region sequence is preferably SEQ ID NO: 25-35, or a sequence substantially identical to any one of the amino acid sequences shown in SEQ ID NOs: 25-35, preferably at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence;

the light chain variable region sequence is preferably SEQ ID NO:36 to 46 or a sequence substantially identical to any one of the amino acid sequences shown in SEQ ID NOs: 36-46, preferably at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence.

In some embodiments, the heavy chain variable region sequence is SEQ ID NO:25; the light chain variable region sequence is SEQ ID NO:36, or a sequence having greater than 85% identity to said sequence; preferably at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence.

In a preferred embodiment, the heavy chain variable region sequence is SEQ ID NO:25; the light chain variable region sequence is SEQ ID NO: 38. 39, 41, 42, 43, 44, or 45, or a sequence having greater than 85% identity to said sequence; preferably at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence.

In a preferred embodiment, the heavy chain variable region sequence is SEQ ID NO:26; the light chain variable region sequence is SEQ ID NO:37, or a sequence having greater than 85% identity to said sequence; preferably at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence.

In a preferred embodiment, the heavy chain variable region sequence is SEQ ID NO: 27. 28, 29, 30, 32, 33, or 34; the light chain variable region sequence is SEQ ID NO:36, or a sequence having greater than 85% identity to said sequence; preferably at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence.

In a preferred embodiment, the heavy chain variable region sequence is SEQ ID NO:31; the light chain variable region sequence is SEQ ID NO:40, or a sequence having greater than 85% identity to said sequence; preferably at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence.

In a further preferred embodiment, the heavy chain variable region sequence is SEQ ID NO:34; the light chain variable region sequence is SEQ ID NO: 38. 42, 43, 44, or 46, or a sequence having greater than 85% identity to said sequence; preferably at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence.

In a further preferred embodiment, the heavy chain variable region sequence is SEQ ID NO:35; the light chain variable region sequence is SEQ ID NO:36. 38, 42 or 46. And more preferably a polypeptide comprising SEQ ID NO:35 and 36, or a sequence having greater than 85% identity to said sequence; preferably at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence.

A polynucleotide encoding the amino acid sequence of any of the above anti-PCSK9 antibodies or antigen-binding fragments, or a sequence at least 85% identical thereto, preferably at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical thereto, or a nucleotide sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conservative nucleotide mutations (preferably substitutions, insertions or deletions) compared to the sequence.

A vector comprising the polynucleotide.

A host cell comprising any one of the antibodies, antigen-binding fragments, polynucleotides, vectors described above. The host cell comprises a prokaryotic cell, a yeast, an insect, or a mammalian cell.

A composition comprising an antibody, antigen-binding fragment, polynucleotide or vector of any one of the above and a pharmaceutically acceptable carrier, diluent or excipient thereof; or optionally in combination with another therapeutic agent. The therapeutic agent includes: a statin drug; the statins include: simvastatin, pitavastatin, mevastatin, atorvastatin, cerivistine, fluvastatin, lovastatin and pravastatin.

A conjugate comprising the fully human anti-PCSK9 antibody or antigen-binding fragment thereof, wherein the conjugate is a detectable label, and the detectable label comprises a fluorescent substance, a luminescent substance, a colored substance, a radioisotope or an enzyme.

Use of an anti-PCSK9 antibody or antigen-binding fragment thereof as described above in the preparation of a reagent for detecting the presence of PCSK9 in a sample to be tested is contacted with any of the anti-PCSK9 antibodies or antigen-binding fragments thereof of the invention, and then the presence of a complex of PCSK9 or fragment thereof with the anti-PCSK9 antibody or antigen-binding fragment thereof is detected, which complex is indicative of the presence of PCSK9.

Use of a conjugate of the fully human anti-PCSK9 antibody or antigen-binding fragment thereof as described above in the preparation of a reagent for detecting the presence of PCSK9 in a sample.

Use of an anti-PCSK9 antibody or antigen-binding fragment thereof as described above in the manufacture of a medicament for the prevention or treatment of a disease or condition that is alleviated, ameliorated, inhibited, or prevented with a PCSK9 antagonist;

wherein the disease or condition comprises hypercholesterolemia, statin intolerance, hyperlipidemia, non-alcoholic fatty liver disease, obesity, diabetes, atherosclerosis, cardiovascular and cerebrovascular diseases, and nephropathy, and the hypercholesterolemia comprises primary hypercholesterolemia, mixed hypercholesterolemia, and familial hypercholesterolemia.

The antibody or antigen binding fragment thereof of the present invention is selected from the following structural forms: full-length antibodies, fab, F (ab') 2, fv, scFv, diabodies, minibodies, diabodies, multispecific antibodies, chimeric antibodies, CDR-grafted antibodies, antibody functional fragments resulting from the fusion of a heavy chain variable region and a complete light chain VH-L and the arrangement, tandem or combination of one or more CDRs.

The antibody or the antibody fragment provides a new medicine for diagnosing, preventing and treating diseases such as cholesterol and lipid metabolic diseases and the like.

Has the advantages that:

(1) The invention adopts a fully human phage antibody library to screen the PCSK 9-targeted antibody, and the obtained antibody is fully human, thereby reducing the immunogenicity to the maximum extent, eliminating the human anti-mouse immune response and prolonging the half life of the antibody drug (Methods, 2005,36 (1): 3-10).

(2) The invention adopts a one-step PCR method, only limited mutation is introduced into key amino acid sites of a CDR region of an antibody, and the efficiency and the success rate of antibody affinity maturation are greatly improved (BMC biotechnology,2008,8 (1): 91 MAbs,2012,4 (6): 664-672).

(3) The full-length antibody Fc segment is modified, so that the affinity to Fc gamma receptors and complement C1q is reduced, and ADCC and CDC cytotoxicity are eliminated (Current Opinion in Biotechnology 2009,20 (6): 685-691).

Drawings

FIG. 1: pTT5-hPCSK9 plasmid map. Amp: an ampicillin resistance gene; PCSK9: a PCSK 9-encoding gene; CMV: a CMV promoter; 6 × His tag: a histidine tag.

FIG. 2:10% (w/v) SDS-PAGE and Western Blot analysis identified purified recombinant human PCSK9 protein.

sds-PAGE analysis to identify purified recombinant human PCSK9 protein, M: protein Marker; lane 1: purified recombinant human PCSK9 protein. Western Blot identifies purified recombinant human PCSK9 proteins.

FIG. 3: phage ELISA detects the relative affinity of the screened anti-human PCSK9 single-chain antibody positive clone and the human PCSK9 protein. Wherein, PCSK9: coating 5 mug/mL human PCSK9; BSA: coating with 5 mug/mL BSA; M13K07: helper phage M13K07 not displaying any single chain antibodies; n =3,means ± SEM.

FIG. 4: pET27b-AP2 plasmid map. Kan: a kanamycin resistance gene; AP2: an AP 2-encoding gene; his tag: a histidine tag.

FIG. 5 is a schematic view of: the parent single chain antibody AP2 was identified by 12% (w/v) SDS-PAGE and Western Blot.

A. Nickel column purified single chain antibody AP2, lane 1: crushing the supernatant; lane 2: purifying the penetration liquid by a nickel column; lane 3: PBS buffer eluent containing 50mM imidazole; lane 4: PBS buffer eluent containing 100mM imidazole; m, protein Marker; lanes 5-8: PBS buffer containing 500mM imidazole.

Western Blot to identify the single-chain antibody AP2.

FIG. 6: amino acid conservation and alternative sequence (preference) analysis of the CDR regions of the parent single chain antibody AP2. After downloading all antibody sequences under the same chothia classification as AP2 in the KABAT database (http:// www. Bio in. Org. Uk/abs/index. Html), amino acid conservation and amino acid preference at each site was analyzed using Weblogo website online mapping: (see: FIGS.: see, et al.)http://weblogo.berkeley.edu/logo.cgi) In the figure, the size of the amino acid letter represents the frequency of occurrence of the amino acid.

FIG. 7 is a schematic view of: phage ELISA detects the relative affinity of the CDR region mutant (AP 2M No. 1-16) of the single-chain antibody AP2 and the human PCSK9 antigen protein. Wherein, PCSK9:5 mug/mL human PCSK9 coating; BSA: coating by 5 mu g/mL BSA; M13K07: helper phage M13K07 not displaying any single chain antibodies; n =4,means ± SEM.

FIG. 8: heavy chain variable region, light chain variable region and CDR region sequences of single-chain antibody AP2 and CDR region mutants (AP 2M No. 1-16). Alignment of antibody sequences and numbering of antibody variable region amino acid sequences according to the Kabat numbering scheme (Kabat numbering scheme) was performed using dnastar, lasergene, v7.1. Analysis software, wherein amino acid residues numbered with letters (a, b, c, etc.) represent the inserted residues defined by the Kabat coding scheme, bold represents CDR regions divided based on the AbM definition (AbM definition), and "-" represents that the amino acid residue at the site is identical to the parent.

FIG. 9: the CDR region mutants (AP 2M Nos. 1 to 16) of the purified single-chain antibody AP2 were identified by 12% (w/v) SDS-PAGE.

A.M: protein Marker; lanes 1-8: CDR region mutants of AP2 (AP 2M Nos. 1 to 8).

B, M: protein Marker; lanes 9-16: CDR region mutants of AP2 (AP 2M Nos. 9 to 16); lane 17: an Ali-scFv positive control (a single-chain antibody in which the heavy chain variable region and the light chain variable region of the antibody Alirocumab are linked via a flexible peptide (G4S) 3).

FIG. 10: effect of the single-chain antibody AP2 and its CDR region mutants (AP 2M No. 1-16) on LDLR protein levels and LDL-C uptake in HepG2 cells.

A. Inhibition of PCSK 9-mediated HepG2 cell surface LDLR degradation, wherein P<0.05vs PCSK9 group； ^# P<0.05vs AP2 group；n＝3,means±SEM。

B. Inhibition of PCSK 9-mediated LDL-C downregulation by HepG2 cells, wherein P<0.05vs PCSK9 group； ^# P<0.05vs AP2 group；n＝3,means±SEM。

FIG. 11: heavy chain variable region, light chain variable region and CDR region sequences of single-chain antibody AP2 and its cross-cloned mutants (AP 2M No. 17-25). Alignment of antibody sequences and numbering of antibody variable region amino acid sequences according to the Kabat numbering scheme (Kabat numbering scheme) was performed using dnastar, lasergene, v7.1. Analysis software, wherein amino acid residues numbered with letters (a, b, c, etc.) represent the inserted residues defined by the Kabat coding scheme, bold represents CDR regions divided based on the AbM definition (AbM definition), and "-" represents that the amino acid residue at the site is identical to the parent.

FIG. 12: cross-cloned mutants of the purified single-chain antibody AP2 (AP 2M No. 17-25) were identified by 12% (w/v) SDS-PAGE. M: protein Marker; lanes 1-9: cross-cloned mutants of the single-chain antibody AP2 (AP 2M Nos. 17 to 25).

FIG. 13 is a schematic view of: effect of the cross-cloned mutants of the single-chain antibody AP2 (AP 2M Nos. 17 to 25) on the LDLR protein level and LDL-C absorption level of HepG2 cells.

A. Inhibition of human PCSK 9-mediated degradation of HepG2 cell surface LDLR, wherein: * P<0.05vs PCSK9 group； ^# P<0.05vs AP2 group；n＝3,means±SEM。

B. Inhibition of human PCSK 9-mediated uptake of LDL-C by HepG2 cells, wherein: * P<0.05vs PCSK9 group； ^# P<0.05vs AP2 group；n＝3,means±SEM。

FIG. 14: western Blot was used to examine the effect of single-chain antibody AP2 cross-cloning mutants (AP 2M No.18, 21) on LDLR protein levels in liver cells of mice as a model of hyperlipidemia after administration. Wherein: * P is<0.05vs Model group；**P<0.01vs Model group； ^# P<0.05vs AP2 group； ^## P<0.01vs AP2group； ^ΔΔ P<0.01vs Normal group；n＝3,means±SEM。

FIG. 15 is a schematic view of: pTT5-AP2/AP2M-HC and pTT5-AP2/AP2M-LC recombinant expression plasmid maps. Amp: an ampicillin resistance gene; AP2/AP2M-HC: an AP2 or AP2M heavy chain encoding gene; AP2/AP2M-LC: an AP2 or AP2M light chain encoding gene; CMV: the CMV promoter.

FIG. 16: the purified full-length anti-human PCSK9 antibody (note: FAP2: the full-length antibody of the parent single-chain antibody AP 2; FAP2M18 and FAP2M21: the full-length antibodies of the single-chain antibody AP2 cross-cloned mutants (AP 2M No.18, 21); alirocumab: the positive control full-length antibody) was examined by 12% (w/v) SDS-PAGE.

A. Non-reducing SDS-PAGE detection: lane M, protein Marker; lanes 1-4: FAP2, FAP2M18, FAP2M21 and Alirocumab. B. Reduced SDS-PAGE detection: lane M, protein Marker; lanes 1-4: FAP2, FAP2M18, FAP2M21 and Alirocumab.

FIG. 17: effect of full-length anti-human PCSK9 antibody on LDLR expression level and LDL-C uptake level of HepG2 cells (Note: FAP2: full-length antibody of parent single-chain antibody AP 2; FAP2M18 and FAP2M21: full-length antibody of single-chain antibody AP2 cross-cloned mutant (AP 2M Nos. 18, 21); alirocumab: positive control full-length antibody).

A. Inhibition of human PCSK 9-mediated degradation of LDLR on the cell surface of HepG2 cells, wherein: * P<0.05vs PCSK9 group；**P<0.01vs PCSK9 group； ^## P<0.01vs Mock group；n＝6,means±SEM。

B. Inhibition of LDL-C uptake by HepG2 cells, wherein: * P<0.01vs PCSK9group； ^### P<0.001vs Mock group；n＝5,means±SEM。

FIG. 18: western Blot was used to examine the effect of the administration of the full-length anti-human PCSK9 antibody into the hepatic cell LDLR protein level in the hyperlipidemia model mice after tail vein administration (note: FAP2M21: full-length antibody which is a single-chain antibody AP2 cross-cloned mutant (AP 2M No. 21); alirocumab: positive control full-length antibody). Wherein: * P<0.05vs Model group；**P<0.01vs Model group；***P<0.001vs Model group； ^#### P<0.0001vs Normal group；n＝3,means±SEM。

Detailed Description

The following examples illustrate specific steps of the present invention, but are not intended to limit the invention.

Terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified.

In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.

Materials, reagents, devices, instruments, apparatuses and the like used in the following examples are commercially available unless otherwise specified.

The invention is further illustrated by the following examples. It will be understood that this example is intended to illustrate the invention and not to limit the scope of the invention in any way.

Example 1 expression purification and characterization of human PCSK9 protein

1.1 construction of recombinant human PCSK9 eukaryotic expression vector

The human PCSK 9-encoding gene (accession No.: NM — 174936.3) was synthesized by seiry biotechnology limited, tokenized to eukaryotic expression vector pTT5, and the recombinant expression plasmid was named pTT5-hPCSK9 (fig. 1), and a 6 × histidine tag (6 × His-tag) was introduced at the 3' end of the PCSK 9-encoding gene in order to facilitate purification of the expression product.

1.2 expression and purification of recombinant human PCSK9 protein

The recombinant human PCSK9 expression plasmid pTT5-hPCSK9 is transfected into CHO3E7 cells by using cationic polymer Polyacetylimine (PEI) for transient expression. Hyclone ^TM Hycell CHO Medium serum-free Medium (GE, cat) ^# SH 30933.02) suspension culture for 7 days, centrifuging, collecting cell culture supernatant, and performing nickel ion affinity column (GE, cat) ^# 17-5318-01) and Superdex 200 size exclusion chromatography (GE, cat) ^# 28-9909-44) to obtain recombinant human PCSK9 protein, and analyzing and identifying the protein by 10% (w/v) SDS-PAGE (figure 2A).

1.3 Western Blot identification of recombinant human PCSK9 protein

Subjecting the purified human PCSK9 protein to SDS-PAGE (separation gel concentration of 10%), transferring at 100V and constant pressure at 4 deg.C for 100min, and transferring the protein to PVDF membrane (Roche, cat) ^# 03010040001 ). The membranes were placed in 5% MTBST (TBS containing 5% skim milk and 0.1% Tween 20) and blocked for 1h at room temperature. Dilution of Anti-PCSK9 rabbit monoclonal antibody (Abcam, cat) with 5% MTBST 1: 3000 ^# ab 181142), incubation at room temperature for 2h, tbst wash 3 times for 10min each. Dilution of HRP-conjugated Goat Anti-Rabbit IgG secondary antibody with 5% MTBST at 1: 5000 (Shanghai Biotech, cat) ^# D110058 Incubation at room temperature for 1h, TBST washing 3 times, each for 10min, ECL color development (FIG. 2B).

Example 2 construction of phage human Single chain antibody library

Construction of phage human single-chain antibody libraries was performed according to the methods described in Nat Biotechnology, 2000,18 (1): 75-80, immunotechnology,1998,3 (4): 271-278. The method uses density gradient centrifugation with 105 parts of healthy human peripheral blood and 58 parts of tumor patient peripheral blood as sourcesThe method comprises the steps of separating peripheral blood lymphocytes generating antibodies, extracting total RNA, carrying out reverse transcription to synthesize cDNA, carrying out PCR amplification by using the cDNA as a template and selecting 6 heavy chain upstream primers, 4 heavy chain downstream primers, 7 lambda chain upstream primers and 3 lambda chain downstream primers of a human specific antibody variable region, adding two enzyme cutting site sequences of Sfi I and Not I and a Linker sequence at two ends of the cDNA, and connecting the heavy chain sequence and the light chain sequence through SOE-PCR to obtain a single-chain antibody scFv fragment. Cloning the single-chain antibody scFv fragment between two enzyme cutting sites Sfi I and Not I of a phagemid vector pCANTAB 5E according to a conventional molecular cloning operation. The ligation product of scFv fragment of single-chain antibody and phagemid pCANTAB 5E was transferred to Escherichia coli TG1 super competent cells (Methods Mol Biol,2004 (248): 117-134) by electrotransformation, and positive colonies were collected to obtain a pool of humanized single-chain antibodies. The final obtained library volume was 1.1X 10 as determined by titer ⁸ The phage human single-chain antibody library of (1).

Example 3 screening of fully human anti-human PCSK9 Single-chain antibodies

3.1 solid phase panning and enrichment of anti-human PCSK9 Single-chain antibodies

Human PCSK9 protein was diluted to 60. Mu.g/mL with a coating buffer (50mM NaHCO3, pH 9.6), and 1mL was added to an immunization tube (Beijing Baokou Weifeian Biotech Co., ltd., cat) ^# 444202 In the rotary shaking table, the solution is coated overnight at 4 ℃ (the coating concentrations of the two, three and four rounds of PCSK9 protein are 30 mu g/mL, 15 mu g/mL and 7.5 mu g/mL respectively). The following day, discard the supernatant, wash the immune tubes rapidly 3 times with PBS, 5% MPBS (PBS containing 5% skim milk) blocked at 37 ℃ for 2h. Discard blocking solution, wash the immune tube rapidly 3 times with PBS, pool activated phage antibody (. About.10) ¹² pfu) was suspended in 4ml2% MPBS and added to the immune tube, and after placing on a rotary shaker and repeated rotation for 2 hours, the immune tube was washed 5 times with PBS containing 0.1% Tween-20, and then washed 5 times with PBS (the number of washing was increased two-fold by one round). 1mL of triethylamine (100 mM) was added, and the incubation was repeated for 10min at room temperature with rotation, and the eluted phage was aspirated and neutralized with 0.5mL of Tris-HCl (1.0M, pH 7.4) and mixed well. After the neutralized phage was infected with 50mL of E.coli TG1 in log phase, the cells were expanded to 200mL, and helper phage M13K07 was added thereto and cultured overnight at 30 ℃. Centrifugal collection the next dayThe culture supernatant was precipitated with PEG/NaCL (20% (w/v) PEG 8000,2.5M NaCl) and used for the next round of panning for 4 rounds.

3.2 Phage ELISA screening of anti-human PCSK9 single-chain antibody

Phage particles eluted in 3-5 rounds are infected with Escherichia coli TG1, and then coated on a plate, and a single colony is picked with a toothpick to a 96-well deep-well plate (Corning Corp., cat) ^# 3590 500. Mu.L of 2 XYT-A (containing 100. Mu.g/mL ampicillin) medium per well was incubated overnight at 37 ℃. 50. Mu.L of the overnight culture per well was plated into a new deep-well plate 2 XYT-AG (containing 100. Mu.g/mL ampicillin and 1% (w/v) glucose) and incubated at 37 ℃ until log phase, as determined by TG1: m13KO7=1:20 of the above-mentioned ratio, the helper phage M13K07 was added, and the mixture was allowed to stand and shake (220 rpm) at 37 ℃ for 30min. After centrifugation at 2000rpm for 10min, the supernatant was aspirated off thoroughly. Each well was resuspended in 500. Mu.L of 2 XYT-AK medium (containing 100. Mu.g/mL ampicillin and 50. Mu.g/mL kanamycin) and incubated at 30 ℃ and 220rpm for about 16 hours. The following day, the deep well plate was centrifuged at 2000rpm for 10min, and the supernatant was the displayed phage antibody for use in the phase ELISA screening.

Human PCSK9 protein was diluted to 5. Mu.g/mL with coating buffer, 100. Mu.L per well was added to a 96-well microplate, and coated overnight at 4 ℃. After sucking out the human PCSK9 coating solution, blocking the solution with 5% MPBS blocking solution at 37 ℃ for 2h, and centrifuging to remove the blocking solution. The monoclonal phage supernatant obtained above was added to a 96-well plate coated with antigen and incubated at 37 ℃ for 2h. The plates were washed 10 times with TBST, and Horseradish Peroxidase (HRP) -labeled anti-M13 antibody (cat, beijing Yiqiao Shenzhou Co., ltd.) was added to each well ^# 11973-MM 05T-H), incubated at 37 ℃ for 1H. Washing the plate 10 times with TBST, adding substrate 3,3', 5' -Tetramethylbenzidine (TMB) for color development, and measuring OD with microplate reader _450nm The value is obtained. The M13KO7 well was used as a negative control, and the positive clones with a higher affinity than three times the absorbance of the negative control were sequenced to obtain 15 wild-type positive clones (pCANTAB 5E-AP1 to 15) expressing the single-chain antibodies AP1 to AP 15, respectively (FIG. 3). The highest affinity AP2 was selected as the parent for further in vitro affinity maturation, and the heavy chain variable region sequence is shown in SEQ ID NO:25, the light chain variable region sequence is shown in SEQ ID NO:36.

example 4 soluble expression and isolation and purification of Single chain antibody AP2

The obtained single-chain antibody AP2 gene was subcloned into expression vector pET27b by conventional molecular cloning procedures to obtain recombinant plasmid pET27b-AP2 (fig. 4), and transformed into e.coli BL21 (DE 3). The recombinant strain was inoculated into 2 XYT-K (containing 50. Mu.g/mL kanamycin) medium and cultured at 37 ℃ to OD _600nm =0.6, add IPTG to final concentration 0.2mM, induce expression for 20h at 16 ℃.4640rpm, centrifuging at 4 deg.C for 10min, and collecting thallus. Periplasmic space protein was extracted by incubation with 0.4mol/L arginine buffer (pH 8.0) at 4 ℃ for 40 min. The mixture was centrifuged at 12000rpm at 4 ℃ for 30min, and the supernatant was collected. Filtering the supernatant with 0.22 μm filter membrane, separating and purifying with nickel column, and eluting with imidazole of different concentrations to remove impurity protein and target protein. 12% (w/v) SDS-PAGE detection of the collected samples (FIG. 5A); the eluate containing the target protein was concentrated by ultrafiltration and subjected to Superdex75 size exclusion chromatography (GE, cat) ^# 17-5174-01) and Anti-6 × His rabbit polyclonal antibody (Shanghai Producer, cat) ^# D110002 Western Blot identification of purified proteins (FIG. 5B).

Example 5 in vitro affinity maturation of anti-human PCSK9 Single-chain antibody AP2

5.1 construction of Secondary mutant library with Single chain antibody AP2 as parent

Numbering of antibody amino acid sequences (numbering) is key to the identification of antibody CDR regions, analysis of amino acid distribution at various sites, and identification of key amino acids (Journal of Molecular Biology,2016,429 (3): 356-364). The present invention employs the most widely used Kabat numbering scheme (Kabat numbering scheme) to number each amino acid position of the heavy chain variable region and the light chain variable region of the single chain antibody AP2 (www.abysis.org), which avoids the inconsistency of the number of amino acids at the same position caused by the conventional numbering method and ensures the consistency of the conserved amino acid positions among different antibodies by introducing the letter designation a, b, c, etc. into the region where the length of the antibody varies (prote: structure, function, and Genetics,1996, 25. Furthermore, the AbM definition (AbM definition) method is preferred in the present invention to partition the CDR regions of single chain antibody AP2, since the AbM definition is the best compromise (compromise) between the Kabat definition (Kabat definition) and the Chothia definition (Chothia definition) and is used by Oxford Molecular's AbM antibody modeling software (Methods in Molecular biology,2004,248 51-91.

Restrictive mutations were introduced into 6 CDR regions of a parent single-chain antibody AP2, respectively, to construct a secondary mutation library, wherein a degenerate oligonucleotide primer was designed to introduce restrictive mutations (Journal of Biotechnology,2015,194 27-36) after analyzing amino acid conservation and mutation status for HCDR1, HCDR2, LCDR1, LCDR2, LCDR3 regions using Kabat database (fig. 6), and a degenerate oligonucleotide primer was designed to introduce random mutations at the hot spot (Journal of Molecular Biology,2011,414 (4): 545-562) after analyzing mutation hot spots for HCDR3 regions (mutatinal hot-spot. The primers used for the AP2 CDR region mutations are shown in table 1, where M = a/C, R = a/G, W = a/T, S = G/C, Y = C/T, K = G/T, V = a/G/C, H = a/C/T, D = a/G/T, B = G/C/T, N = a/G/C/T.

TABLE 1 primers used for mutating each CDR region of AP2

Note: bold + underline indicates base mutation sites

Using the recombinant plasmid pCANTAB 5E-AP2 encoding the parent single-chain antibody AP2 of example 3.2 as a template, the fast high fidelity enzyme PrimeSTAR Max DNA Polymerase (Takara, cat) ^# R045A) and each CDR area primer respectively carry out full plasmid PCR amplification on 6 CDR areas, and after the recovery of PCR amplification product gel, the PCR amplification product gel is subjected to Dpn I enzyme digestion and ethanol precipitation and then is electrically transformed into Escherichia coli E.coli TG1 super competent cells, namely the mutant secondary library. Accumulating the library capacity through multiple times of electric transformation to make the library capacity of the mutant reach 4.2 multiplied by 10 ⁷ 。

5.2 biopanning and Positive clone screening of Secondary mutant pools

Biopanning and positive clone screening were performed as in example 3 (fig. 7), and clones with higher affinity were sequenced to obtain 16 CDR region mutant single-chain antibodies of AP2, designated AP2M1, AP2M2, AP2M3, AP2M4, AP2M5, AP2M6, AP2M7, AP2M8, AP2M9, AP2M10, AP2M11, AP2M12, AP2M13, AP2M14, AP2M15, and AP2M16, respectively. The amino acid sequence of the mutated CDR region is shown in FIG. 8.

5.3 determination of affinity constants of Single-chain antibody AP2 and CDR region mutants thereof

Single-chain antibody AP2 and its CDR region mutants AP2M 1-16 proteins were expressed and purified according to the method of example 4, and 12% (w/v) SDS-PAGE was identified as shown in FIG. 9. Using ForteBio Octet QK ^e A biomacromolecule interaction analyzer is used for measuring the affinity constants of AP2 and mutant AP2M 1-16 proteins in CDR regions thereof and human PCSK9, and the basic process is as follows: human PCSK9 protein and biotin are mixed in a molar ratio of 1:5, uniformly mixing, incubating at room temperature for 2h, and removing unreacted biotin by using a G25 column to obtain the biotinylated human PCSK9. The instrument was turned on earlier for more than 45min before the assay, and the biosensor was placed in a1 XSD buffer (PBS, pH7.4,0.02% Tween 20,0.1% BSA) and hydrated (Hydrate) for at least 10min. In black 96-well plates (Corning Corp., cat) ^# MH 0255) at column 1 and column 3 200 μ L SD Buffer was added per well. Biotinylated human PCSK9 was diluted to 50 μ g/mL with SD Buffer and added to column 2 at 200 μ L per well. The single-stranded antibody to be tested was diluted with SD Buffer from 6000nM or 3000nM for at least 4 concentration gradients and added to column 4 at 200. Mu.L per well, with SD Buffer added to the last well of the column as a control well. Place 96-well plate in Octet QK ^e In the biomacromolecule interaction instrument, a running program is set, and a Streptavidin biosensor (Streptavidin biosensors) is balanced in an SD buffer for 60s, biotinylated PCSK9 for 300s and in the SD buffer for 120s, then combined with a single-chain antibody for 300s and dissociated in the SD buffer for 600s. Using ForteBio Octet QK ^e Data analysis software calculated equilibrium dissociation constant KD values and other kinetic parameters. The results (Table 2) show that the single-chain antibody AP2 and the CDR region mutants AP2M1 to AP 16 can be combined with PCSK9.

TABLE 2 binding Rate constant (kon), dissociation Rate constant (koff) and equilibrium dissociation constant (KD) for the Single chain antibody AP2 and its CDR region mutants to PCSK9

Serial number	clones	kon(M ^-1 s ^-1 )	koff(s ^-1 )	KD(M)
					1	AP2	1.41E+04	1.23E-03	8.72E-08
2	AP2M1	3.52E+05	6.08E-03	1.73E-08
					3	AP2M2	1.01E+04	1.04E-03	1.03E-07
4	AP2M3	4.59E+04	2.23E-03	4.87E-08
					5	AP2M4	1.36E+05	4.38E-02	3.22E-07
6	AP2M5	7.02E+04	2.11E-03	3.00E-08
					7	AP2M6	1.44E+05	3.49E-03	2.41E-08
8	AP2M7	6.12E+04	7.89E-03	1.29E-07
					9	AP2M8	5.40E+04	2.90E-03	5.37E-08
10	AP2M9	9.41E+04	5.39E-03	5.73E-08
					11	AP2M10	1.27E+05	3.32E-03	2.62E-08
12	AP2M11	7.03E+02	8.23E-02	1.17E-04
					13	AP2M12	9.14E+04	2.28E-03	2.50E-08
14	AP2M13	4.67E+04	8.30E-04	1.78E-08
					15	AP2M14	5.72E+04	2.80E-03	4.89E-08
16	AP2M15	1.19E+04	6.81E-04	5.72E-08
					17	AP2M16	6.48E+04	1.43E-02	2.21E-07
18	Ali-scFv	8.93E+03	4.55E-04	5.09E-08

Example 6 in vitro Activity assay of Single chain antibody AP2 and its CDR region mutants

6.1 inhibition of PCSK 9-mediated LDLR degradation by Single-chain antibody AP2 and CDR region mutants thereof

HepG2 cells were cultured to logarithmic phase, 5X 10% in 10% FBS MEM medium under conditions of 37 ℃ and 5% CO2% ⁵ cells/well were plated in 6-well cell culture plates at 500. Mu.L/well and incubated overnight. The next day, the original medium was changed to opti-MEM medium without plasma and incubation was continued for 12h. HepG2 cells were divided into 3 groups: group 1 was negative control (Mock group); group 2 added PCSK9 protein at a final concentration of 20 μ g/mL; the PCSK9 protein and the anti-PCSK9 single-chain antibody AP2 or a CDR region mutant (AP 2M 1-16) or Ali-scFv were added to the remaining administration groups at a final concentration of 20. Mu.g/mL and 50. Mu.g/mL, respectively. The culture was continued at 37 ℃ for 12h.

The cells are digested from a 6-well plate, an appropriate amount of RIPA lysis buffer is added for ice lysis for 30min, the cells are centrifuged at 12000rpm for 10min, and the supernatant is used for Western Blot analysis to detect the change of total LDLR of HepG2 cells. Wherein the primary antibody is Rabbit Anti-LDL Receptor antibody (Abcam, cat) ^# ab 52818), secondary antibody was HRP-conjugated coat Anti-Rabbit IgG (cat, shanghai Biotech) ^# D110058)。

The results (fig. 10A) show that PCSK9 can effectively promote the degradation of LDLR on HepG2 cell surface, while the single-chain antibody AP2 and its CDR region mutants (AP 2M 1-16) can inhibit the degradation of PCSK9 on HepG2 cell LDLR to different degrees.

6.2 Effect of Single-chain antibody AP2 and CDR region mutants thereof on LDL-C uptake by HepG2 cells

After treating the cells with the single-chain antibody AP2 and its CDR region mutants (AP 2M 1-16) for 12h as described in 6.1, 1.5. Mu.g of DiI-LDL (Guangzhou Yiyuanzhi Bio Inc., cat) was added to each well ^# YB-0011), continuously culturing for 4h, washing with PBS for two times, and detecting the fluorescence intensity (excitation wavelength of 520nm and emission wavelength of 580 nm) of each hole by using a full-wavelength microplate reader. The change in LDL uptake activity of HepG2 cells after treatment with different drugs was compared against HepG2 cells treated without human PCSK9 protein (fig. 10B).

By comparing the affinity and the biological activity, 6 CDR region mutant single-chain antibodies of AP2 with better affinity and biological activity are obtained: AP2M3, AP2M10, AP2M12, AP2M13, AP2M14, AP2M15. The Heavy Chain Variable Region (HCVR) sequence and the Light Chain Variable Region (LCVR) sequence are shown in FIG. 11.

Example 7 CDR region mutants of the Single chain antibody AP2 further affinity maturation in vitro

7.1 Cross-cloning of CDR region mutants of the Single chain antibody AP2

CDR regions of each mutant derived from the same parent antibody are subjected to cross cloning (cross-cloning) by adopting an overlap extension PCR method (Molecular Immunology,2008,46 (1): 135-144) so as to obtain an anti-human PCSK9 single-chain antibody with better affinity and activity. Since the CDR region mutant single-chain antibodies of AP2 have the same framework regions, primers (table 3) can be designed using the framework region sequences as templates, and CDR regions of the CDR region mutant single-chain antibodies of AP2 (AP 2M3, AP2M10, AP2M12, AP2M13, AP2M14, and AP2M 15) having the strains of example 6 with better affinity and biological activity can be cross-cloned (cross-cloning) by overlap extension PCR (overlap PCR). For example, AP2M13 (HCDR 1) and AP2M12 (LCDR 1) were combined by overlap extension PCR to obtain a novel single-chain antibody (named AP2M 17) with the heavy chain variable region sequence shown in SEQ ID No.34 and the light chain variable region sequence shown in SEQ ID No.42. 9 strains of cross-cloned (cross-cloned) mutant antibody were obtained according to this method (FIG. 11).

TABLE 3 primers for CDR region mutant cross-cloning of the Single-chain antibody AP2

7.2 Cross-clone mutant antibody affinity constant determination

The 9-strain cross-cloned antibody of FIG. 11 was expressed and purified according to the method of example 4, and its purity was identified by 12% (w/v) SDS-PAGE (FIG. 12). The affinity constant of the recombinant human PCSK9 protein was determined by the method of example 5.3, and the results are shown in table 4.

TABLE 4 binding Rate constant (kon), dissociation Rate constant (koff) and equilibrium dissociation constant (KD) of Cross-clonal mutant antibodies to PCSK9

Serial number	Cross-cloned antibodies	kon(M ^-1 s ^-1 )	koff(s ^-1 )	KD(M)
					1	AP2M17	7.85E+04	1.14E-03	1.46E-08
2	AP2M18	1.08E+04	5.41E-04	5.03E-08
					3	AP2M19	8.98E+05	1.07E-02	1.18E-08
4	AP2M20	5.31E+04	1.51E-03	2.84E-08
					5	AP2M21	1.29E+05	4.67E-04	3.61E-09
6	AP2M22	4.54E+04	6.13E-04	1.35E-08
					7	AP2M23	9.58E+04	4.15E-03	4.33E-08
8	AP2M24	1.85E+05	1.91E-03	1.03E-08
					9	AP2M25	4.17E+04	1.85E-03	4.43E-08

7.3 determination of biological Activity of Cross-Linked mutant antibodies

The effect of cross-clonal mutant antibody treatment on LDLR levels and LDL-C uptake in HepG2 cells was examined as described in example 6. The results show that PCSK 9-mediated degradation of LDLR was significantly inhibited after treatment of HepG2 cells with the cross-clonal mutant antibody (fig. 13A), while LDL-C uptake by HepG2 cells was significantly upregulated (fig. 13B). Among them, AP2M18 and AP2M21 had the highest activity.

7.4 measurement of hypolipidemic Activity in Cross-cloned mutant antibodies AP2M18 and AP2M21 mice

C57BL/6 male mice were acclimatized for one week, randomly divided into a normal group, a model group, and 4 administration groups (AP 2 group, AP2M18 group, AP2M21 group, and Ali-scFv group), and n =6. Human PCSK9 expression plasmid pTT5-hPCSK9 (50. Mu.g in 2mL saline) was injected intravenously in the 5s internal tail vein and human PCSK9 was overexpressed in mice, and normal controls were injected with saline as described in the literature (GENE THERAPY,1999,6 (7): 1258-1266). On day 6 of model building, mice in the administration group were administered a single chain antibody (10 mg/kg in 200. Mu.L PBS) by intraperitoneal injection, and the model group and the normal control group were administered an equal volume of PBS. After administration, mice were fasted and fed without water for 18h, the eyeball was picked the next day to collect blood and liver, the blood sample was placed in a refrigerator at 4 ℃ for 4h, centrifuged at 4000rpm for 15min, the upper serum was taken, and LDL-C detection kit (Nanjing institute of bioengineering, cat) ^# A113-1) and TC detectionKit (Nanjing institute of bioengineering, cat) ^# A111-1) measuring the levels of LDL-C and TC in serum. Meanwhile, after the liver of the mouse is homogenized and treated by RIPA lysate, the protein levels of LDLR and PCSK9 in each group are detected by Western Blot. The results indicate that AP2M21 significantly reduced plasma LDL-C (39.04%) and TC (26.01%) (table 5) and significantly inhibited PCSK 9-mediated LDLR degradation (figure 14), with activity comparable to the positive control Ali-scFv.

TABLE 5 Effect of anti-PCSK9 Single-chain antibodies on plasma Cholesterol LDL-C, TC levels in hyperlipidemic model mice

"n" indicates the number of animals, "###" indicates that P is <0.01 compared to a normal control group; "," indicates P <0.05, 0.01 (n =6, values are means ± SEM) compared to the model control group.

Example 8 full-Length antibody construction and bioactivity analysis

8.1 full Length antibody preparation

The conventional molecular biology method is adopted to construct the full-length antibody of the parent single-chain antibody AP2 and the cross-cloning mutant antibodies AP2M18 and AP2M21. The specific process is as follows: the VL sequence was ligated to the lambda-type light chain constant region (SEQ NO. 47), the VH sequence was ligated to the IgG1 heavy chain constant region (SEQ NO. 48) and subcloned into the eukaryotic expression vector pTT5, respectively (FIG. 15). Simultaneously, a Kozak sequence (GCCGCCACC) and a signal peptide sequence are introduced into the N ends of VH and VL. In addition, in order to reduce ADCC and CDC effects, amino acids 234 and 235 of the heavy chain constant region sequence were mutated to Ala, amino acid 297 to Gly, and C-terminal Lys was removed to improve the uniformity of the expression product of the full-length antibody (Cellular Immunology 2000,200 (1): 16-26; journal of biological chemistry 2017,292 (5): 1865-1875; journal of Pharmaceutical sciences,2016,105 (7): 2066-2072). Meanwhile, the positive control antibody Alirocumab heavy chain and light chain genes were cloned into the expression vector pTT5, respectively.

The heavy chain and light chain expression plasmids are co-transfected into CHO3E7 cells by adopting a PEI transfection method, cell culture liquid supernatant is collected after 7 days of serum-free culture medium culture, and after affinity chromatography and purification of protein A column (Roche, cat # 11134515001), anti-PCSK9 full-length antibodies (shown in figure 16) corresponding to AP2, AP2M18 and AP2M21 are obtained and are respectively named as FAP2, FAP2M18 and FAP2M21.

8.2 detection of full-Length antibody Activity in vitro

The effects of full-length antibodies FAP2, FAP2M18 and FAP2M21 on LDLR protein levels and LDL-C uptake function of HepG2 cells were examined as described in example 6. The results show that the full-length antibody FAP2M21 can significantly inhibit the degradation of PCSK9 to LDLR (FIG. 17A) and significantly enhance the LDL-C absorption function of HepG2 cells (FIG. 17B), and the activity of the full-length antibody is equivalent to that of positive control Alirocumab.

8.3 Fortebio OctetQK ^e Determination of full-Length antibody affinity constants

The affinity constants of FAP2M21 and its parent antibody FAP2 and positive control Alirocumab to human PCSK9 were determined as in example 5.3 with antibody concentration gradients set at 800nM,400nM,200nM and 100nM and antibody affinity constants were determined by flow over the SA sensor surface of immobilized (Immobilize) human PCSK9 protein, the results are shown in Table 6.

TABLE 6 anti-PCSK9 full-length antibody affinity constants

Serial number	anti-PCSK9 antibodies	kon(M ^-1 s ^-1 )	koff(s ^-1 )	KD(M)
					1	FAP2	1.76E+05	5.76E-03	3.28E-08
2	FAP2M21	3.33E+03	4.68E-06	1.41E-09
					3	Alirocumab	8.04E+04	6.87E-05	8.54E-10

8.4 detection of hypolipidemic Activity in anti-PCSK9 full-length antibody mice

The hyperlipemia mouse model was established as in example 7.4, and 48C 57BL/6 male mice were divided into 8 groups (n = 6) in total, and 1 group of each of the normal control and model groups and 6 groups of the administered group were set. Tail vein injection (administration volume 200. Mu.L) was started on day 6 after molding, physiological saline was administered to the normal group and the model group, and positive control agents Alirocumab and FAP2M21 antibody were administered at low, medium and high doses (1,3, 10mg/kg, respectively).

After administration, mice were fasted and kept without water for 18h, and blood and liver were collected from the next day by eye picking, and LDL-C and TC levels in the mice serum were measured as described in example 7.4, while PCSK9 and LDLR protein levels in liver tissues were measured by Western Blot. The results show that both FAP2M21 and Alirocumab significantly inhibited PCSK 9-mediated degradation of LDL-R (figure 18), while dose-dependently reduced LDL-C and TC levels in mouse plasma (table 7). Compared with a model group, the LDL-C levels of the FAP2M21 antibody in the low, medium and high dose administration groups are respectively reduced by 3.3%, 30.2% and 37.2%, the TC level is reduced by 5.2%, 12.8% and 16.7%, and the hypolipidemic activity of the FAP2M21 antibody is equivalent to that of a positive control drug Alirocumab.

TABLE 7 Effect of anti-PCSK9 full-length antibody on serum LDL-C, TC levels in hyperlipidemic model mice

"n" indicates the number of animals, "####" indicates that P is <0.001 compared to the normal control group; "," indicates P <0.05, 0.01 (n =6, values are means ± SEM) compared to the model control group.

Sequence listing

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<213> Homo sapiens

<400> 12

Arg Ala Val Gly Gln Gly Ser Glu Tyr

1 5

<210> 13

<211> 13

<212> PRT

<213> Homo sapiens

<400> 13

Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Thr Val Asn

1 5 10

<210> 14

<211> 13

<212> PRT

<213> Homo sapiens

<400> 14

Ser Gly Ser Ser Ser Asn Ile Gly Asn Pro Trp Val Phe

1 5 10

<210> 15

<211> 13

<212> PRT

<213> Homo sapiens

<400> 15

Ser Gly Ser Ser Ser Asn Ile Gly Ser Ser Glu Val Phe

1 5 10

<210> 16

<211> 7

<212> PRT

<213> Homo sapiens

<400> 16

Arg Asn Asn Gln Arg Pro Ser

1 5

<210> 17

<211> 11

<212> PRT

<213> Homo sapiens

<400> 17

Ala Ala Trp Asp Asp Ser Leu Ser Ala Arg Val

1 5 10

<210> 18

<211> 11

<212> PRT

<213> Homo sapiens

<400> 18

Ala Tyr Trp Asp Asp Thr Leu Ser Ala Arg Leu

1 5 10

<210> 19

<211> 11

<212> PRT

<213> Homo sapiens

<400> 19

Ser Ser Trp Asp Pro Ser Leu Leu Leu Gly Val

1 5 10

<210> 20

<211> 11

<212> PRT

<213> Homo sapiens

<400> 20

Asp Ser Trp Asp His Ser Leu Ser Ser Met Leu

1 5 10

<210> 21

<211> 11

<212> PRT

<213> Homo sapiens

<400> 21

Ala Ser Trp Asp Asp Cys Leu Ser Ala Arg Pro

1 5 10

<210> 22

<211> 11

<212> PRT

<213> Homo sapiens

<400> 22

Ala Ile Trp Asp Asp His Leu Ser Ala Arg Asn

1 5 10

<210> 23

<211> 11

<212> PRT

<213> Homo sapiens

<400> 23

Asn Val Trp Asp Pro Ser Leu Arg Arg Gly Leu

1 5 10

<210> 24

<211> 11

<212> PRT

<213> Homo sapiens

<400> 24

Ser Ser Trp Asp Ser Ser Leu Ser Arg Ala Val

1 5 10

<210> 25

<211> 118

<212> PRT

<213> Homo sapiens

<400> 25

Gln Val Thr Leu Lys Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ala Val Gly Gln Gly Trp Arg Phe Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 26

<211> 118

<212> PRT

<213> Homo sapiens

<400> 26

Gln Val Thr Leu Lys Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ala Val Gly Gln Gly Ser Leu Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 27

<211> 118

<212> PRT

<213> Homo sapiens

<400> 27

Gln Val Thr Leu Lys Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ala Val Gly Gln Gly Tyr His Thr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 28

<211> 118

<212> PRT

<213> Homo sapiens

<400> 28

Gln Val Thr Leu Lys Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Glu Phe Ser Asp Tyr

20 25 30

Phe Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ala Val Gly Gln Gly Trp Arg Phe Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 29

<211> 118

<212> PRT

<213> Homo sapiens

<400> 29

Gln Val Thr Leu Lys Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Glu Phe Arg Arg Tyr

20 25 30

Thr Leu Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ala Val Gly Gln Gly Trp Arg Phe Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 30

<211> 118

<212> PRT

<213> Homo sapiens

<400> 30

Gln Val Thr Leu Lys Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Glu Ile Arg Arg Tyr

20 25 30

Glu Phe Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ala Val Gly Gln Gly Trp Arg Phe Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 31

<211> 118

<212> PRT

<213> Homo sapiens

<400> 31

Gln Val Thr Leu Lys Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ala Val Gly Gln Gly Ser Glu Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 32

<211> 118

<212> PRT

<213> Homo sapiens

<400> 32

Gln Val Thr Leu Lys Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Glu Lys Ser Ser Glu Ser Arg Ser Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ala Val Gly Gln Gly Trp Arg Phe Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 33

<211> 118

<212> PRT

<213> Homo sapiens

<400> 33

Gln Val Thr Leu Lys Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Cys Ile Phe Leu Ile Tyr

20 25 30

Glu Cys Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ala Val Gly Gln Gly Trp Arg Phe Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 34

<211> 118

<212> PRT

<213> Homo sapiens

<400> 34

Gln Val Thr Leu Lys Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Asn Phe Asn Trp Tyr

20 25 30

Glu Leu Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ala Val Gly Gln Gly Trp Arg Phe Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 35

<211> 118

<212> PRT

<213> Homo sapiens

<400> 35

Gln Val Thr Leu Lys Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Asn Phe Asn Trp Tyr

20 25 30

Glu Leu Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Glu Lys Ser Ser Glu Ser Arg Ser Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ala Val Gly Gln Gly Trp Arg Phe Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 36

<211> 111

<212> PRT

<213> Homo sapiens

<400> 36

Ser Tyr Glu Leu Thr Gln Leu Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn

20 25 30

Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Asp Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Ser Ala Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 37

<211> 111

<212> PRT

<213> Homo sapiens

<400> 37

Ser Tyr Glu Leu Thr Gln Leu Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn

20 25 30

Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Asp Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Tyr Trp Asp Asp Thr Leu

85 90 95

Ser Ala Arg Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 38

<211> 111

<212> PRT

<213> Homo sapiens

<400> 38

Ser Tyr Glu Leu Thr Gln Leu Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn

20 25 30

Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Asp Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Trp Asp Pro Ser Leu

85 90 95

Leu Leu Gly Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 39

<211> 111

<212> PRT

<213> Homo sapiens

<400> 39

Ser Tyr Glu Leu Thr Gln Leu Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn

20 25 30

Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Asp Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Asp Ser Trp Asp His Ser Leu

85 90 95

Ser Ser Met Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 40

<211> 111

<212> PRT

<213> Homo sapiens

<400> 40

Ser Tyr Glu Leu Thr Gln Leu Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn

20 25 30

Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Asp Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Trp Asp Asp Cys Leu

85 90 95

Ser Ala Arg Pro Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 41

<211> 111

<212> PRT

<213> Homo sapiens

<400> 41

Ser Tyr Glu Leu Thr Gln Leu Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Pro

20 25 30

Trp Val Phe Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Asp Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Ser Ala Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 42

<211> 111

<212> PRT

<213> Homo sapiens

<400> 42

Ser Tyr Glu Leu Thr Gln Leu Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Ser

20 25 30

Glu Val Phe Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Asp Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Ser Ala Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 43

<211> 111

<212> PRT

<213> Homo sapiens

<400> 43

Ser Tyr Glu Leu Thr Gln Leu Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn

20 25 30

Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Asp Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ile Trp Asp Asp His Leu

85 90 95

Ser Ala Arg Asn Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 44

<211> 111

<212> PRT

<213> Homo sapiens

<400> 44

Ser Tyr Glu Leu Thr Gln Leu Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn

20 25 30

Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Asp Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Val Trp Asp Pro Ser Leu

85 90 95

Arg Arg Gly Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 45

<211> 111

<212> PRT

<213> Homo sapiens

<400> 45

Ser Tyr Glu Leu Thr Gln Leu Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn

20 25 30

Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Asp Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Trp Asp Ser Ser Leu

85 90 95

Ser Arg Ala Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 46

<211> 111

<212> PRT

<213> Homo sapiens

<400> 46

Ser Tyr Glu Leu Thr Gln Leu Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Ser

20 25 30

Glu Val Phe Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Asp Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Trp Asp Pro Ser Leu

85 90 95

Leu Leu Gly Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 47

<211> 105

<212> PRT

<213> Homo sapiens

<400> 47

Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu

1 5 10 15

Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe

20 25 30

Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val

35 40 45

Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys

50 55 60

Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser

65 70 75 80

His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu

85 90 95

Lys Thr Val Ala Pro Thr Glu Cys Ser

100 105

<210> 48

<211> 329

<212> PRT

<213> Homo sapiens

<400> 48

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly

325

Claims

1. A fully human anti-PCSK9 antibody or antigen-binding fragment thereof, characterized in that: comprising a heavy chain variable region and a light chain variable region,

the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO:35, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO 36.

2. The fully human anti-PCSK9 antibody or antigen-binding fragment thereof of claim 1, which is a full-length antibody, scFv, fab, F (ab') 2, fv, diabody, or multispecific antibody.

3. A polynucleotide encoding the fully human anti-PCSK9 antibody or antigen-binding fragment thereof of claim 1.

4. A vector comprising the polynucleotide of claim 3.

5. A host cell comprising the fully human anti-PCSK9 antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, the polynucleotide according to claim 3, and the vector according to claim 4.

6. A composition comprising the fully human anti-PCSK9 antibody or antigen-binding fragment thereof of any of claims 1-2, the polynucleotide of claim 3, the vector of claim 4, and a pharmaceutically acceptable carrier, diluent, or excipient thereof.

7. The composition of claim 6, wherein it is combined with another therapeutic agent.

8. Use of the fully human anti-PCSK9 antibody or antigen-binding fragment thereof of claim 1 in the preparation of a reagent for detecting the presence of PCSK9 in a sample.

9. Use of the fully human anti-PCSK9 antibody or antigen-binding fragment thereof of claim 1 in the manufacture of a medicament for the treatment of hyperlipidemia.

10. The use according to claim 9, characterized in that the hyperlipidemia comprises hypercholesterolemia, including primary hypercholesterolemia, mixed hypercholesterolemia, and familial hypercholesterolemia.