Fully human anti-PCSK9 antibody, antigen-binding fragment thereof and application thereof
阅读说明：本技术 全人源抗pcsk9抗体、其抗原结合片段及其应用 (Fully human anti-PCSK9 antibody, antigen-binding fragment thereof and application thereof ) 是由 谭树华 徐梦龙 汪可 陈满满 雷高新 于 2020-06-16 设计创作，主要内容包括：本发明公开了全人源抗PCSK9抗体或其抗原结合片段及其应用,本发明以人PCSK9为抗原从高容量全人源噬菌体抗体库中筛选出抗PCSK9单链抗体。然后,采用体外亲和力成熟、CDR区交叉克隆及抗体全长化技术获得新型、高活性、全人源抗PCSK9单链及全长抗体。上述抗体及其片段可特异性结合PCSK9,有效抑制PCSK9介导的肝细胞表面低密度脂蛋白受体(LDLR)的降解,显著增强肝细胞对低密度脂蛋白胆固醇(LDL-C)的摄取,从而明显降低血清LDL-C水平。本发明所述的抗体及其片段科应用于制备预防和治疗PCSK9介导的包括高胆固醇血症、高血脂症肥胖症、动脉粥样硬化以及心脑血管疾病等相关疾病的药物。(The invention discloses a fully human anti-PCSK9 antibody or an antigen binding fragment thereof and application thereof, and in the invention, an 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 low-density lipoprotein receptor (LDLR) on the surface of a liver cell, and remarkably enhance the uptake of low-density lipoprotein cholesterol (LDL-C) by the liver cell, thereby obviously reducing the level of serum LDL-C. The antibody and the fragment thereof are applied to preparing medicines for preventing and treating PCSK 9-mediated diseases related to hypercholesterolemia, hyperlipidemia obesity, atherosclerosis, cardiovascular and cerebrovascular diseases and the like.)
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 heavy chain variable region comprises complementarity determining regions HCDR1, HCDR2, HCDR 3; the light chain variable region comprises complementarity determining regions LCDR1, LCDR2, LCDR 3;
wherein:
HCDR1 comprises SEQ ID NO: 1-6, or any amino acid sequence as set forth in SEQ ID NO: 1-6, wherein the amino acid sequences have at least 85% identity; and/or
HCDR2 comprises SEQ ID NO: 7. 8, or comprises an amino acid sequence identical to any one of SEQ ID NOs: 7. 8, or a sequence having at least 85% identity to any one of the amino acid sequences set forth in seq id no; and/or
HCDR3 comprises SEQ ID NO: 9-12, or any amino acid sequence shown in SEQ ID NO: 9-12, wherein any one of the amino acid sequences is at least 85% identical; and/or
LCDR1 comprises SEQ ID NO: 13-15, or any amino acid sequence as set forth in SEQ ID NO: 13-15, wherein any one of the amino acid sequences shown in seq id No. 13 has at least 85% identity; and/or
LCDR2 comprises SEQ ID NO: 16 or comprises an amino acid sequence substantially identical to SEQ ID NO: 16 has at least 85% identity to the amino acid sequence set forth in seq id no; and/or
LCDR3 comprises SEQ ID NO: 17-24, or any amino acid sequence as set forth in SEQ ID NO: 17-24, wherein any one of the amino acid sequences shown in seq id No.17 has at least 85% identity.
2. The fully human anti-PCSK9 antibody or antigen-binding fragment thereof according to claim 1, wherein the sequence of the heavy chain variable region comprises SEQ ID NO: 25-35, or any amino acid sequence as set forth in SEQ ID NO: 25-35, wherein any amino acid sequence has 85% identity or more; and/or
The light chain variable region sequence comprises SEQ ID NO: 36-46, or a sequence comprising an amino acid sequence substantially identical to seq id NO: 36 to 46, and a sequence having 85% or more identity to any one of the amino acid sequences shown in FIGS.
3. The fully human anti-PCSK9 antibody or antigen-binding fragment thereof according to claim 2, wherein the antibody or antigen-binding fragment comprises any of:
(a) an antibody or antigen-binding fragment comprising the heavy chain variable region set forth in SEQ ID NO 25 and the light chain variable region set forth in any of SEQ ID NOs 36, 38, 39, 41, 42, 43, 44, or 45, or a sequence having at least 85% identity thereto;
(b) an antibody or antigen-binding fragment comprising the heavy chain variable region of SEQ ID NO 26 and the light chain variable region of SEQ ID NO 37, or a sequence having at least 85% identity thereto;
(c) an antibody or antigen-binding fragment comprising the heavy chain variable region of SEQ ID NO 27, 28, 29, 30, 32, 33 or 34 and the light chain variable region of SEQ ID NO 36, or a sequence having at least 85% identity thereto;
(d) an antibody or antigen-binding fragment comprising the heavy chain variable region shown as SEQ ID NO 31 and the light chain variable region shown as SEQ ID NO 40, or a sequence having at least 85% identity thereto;
(e) an antibody or antigen-binding fragment comprising the heavy chain variable region shown as SEQ ID NO 34 and the light chain variable region shown as SEQ ID NOs 38, 42, 43, 44 or 46, or a sequence having at least 85% identity thereto;
(f) an antibody comprising the heavy chain variable region shown as SEQ ID NO 35 and the light chain variable region shown as SEQ ID NOs 36, 38, 42 or 46, or sequences having at least 85% identity thereto.
4. The fully human anti-PCSK9 antibody or antigen-binding fragment thereof according to claim 1, characterized in that it is a full-length antibody, scFv, Fab, F (ab') 2, Fv, diabody, multispecific antibody or any stable such fragment.
5. A polynucleotide encoding the fully human anti-PCSK9 antibody or antigen-binding fragment thereof of any of claims 1-4.
6. A vector comprising the polynucleotide of claim 5.
7. A host cell comprising the fully human antibody, antigen-binding fragment, polynucleotide, vector of any one of claims 1-6.
8. A composition comprising a fully human antibody, antigen-binding fragment, polypeptide, polynucleotide or vector of any one of claims 1-6 and a pharmaceutically acceptable carrier, diluent or excipient thereof; or optionally in combination with another therapeutic agent.
9. A conjugate comprising the fully human anti-PCSK9 antibody or antigen-binding fragment thereof of claims 1-3, wherein the conjugate is a detectable label comprising a fluorescent substance, a luminescent substance, a colored substance, a radioisotope, or an enzyme.
10. Use of the fully human anti-PCSK9 antibody or antigen-binding fragment thereof of any one of claims 1 to 3 in the preparation of a reagent for detecting the presence of PCSK9 in a sample.
11. Use of the conjugate of claim 9 comprising the fully human anti-PCSK9 antibody or antigen-binding fragment thereof of claims 1-3 in the preparation of a reagent for detecting the presence of PCSK9 in a sample.
12. Use of the fully human anti-PCSK9 antibody or antigen-binding fragment thereof of any of claims 1-3 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.
13. The use according to claim 12, wherein the disease or condition comprises hypercholesterolemia, statin intolerance, hyperlipidemia, non-alcoholic fatty liver disease, obesity, diabetes, atherosclerosis, cardiovascular and cerebrovascular diseases, nephropathy, the hypercholesterolemia comprises primary hypercholesterolemia, mixed hypercholesterolemia, and familial hypercholesterolemia.
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to a novel fully human Proprotein convertase subtilisin/kexin 9 (PCSK 9) resisting 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: 17093). 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 lysosome, 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 that is expressed predominantly 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; 32(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 in lysosomes, 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 and the development of hypercholesterolemia in the blood (Proc Natl Acad Sci U S A.2008; 105(35): 13045-13050; circulation research.2014; 114(6): 1036). Therefore, drugs that inhibit the synthesis and secretion of PCSK9 and block its interaction with LDLR can promote LDL-C clearance and reduce the risk of hypercholesterolemia (Annu Rev Pharmacol Toxicol.2014; 54: 273-293).
In addition, it has been reported in the literature that PCSK9 elevation is closely related to obesity and
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, USA (AMG145) and the fully human monoclonal antibody Alirocumab developed by Regeneron/Sanofi, REGN727/SAR236553, have been approved in the United states and Europe for marketing (J Clin Pharmacol.2017; 57(1):7-32) for the treatment of heterozygous familial hypercholesterolemia, or the treatment of cardiovascular high risk patients for whom 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 an 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 (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 the like.
Disclosure of Invention
The purpose of the invention is as follows: 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 be specifically bound with PCSK9, and effectively inhibit degradation of PCSK9 to LDLR on the surface of hepatocytes, thereby increasing the level of LDLR of hepatocytes and enhancing the absorption function of hepatocytes to LDL-C.
The invention also aims to solve the technical problem of providing application of the fully human anti-PCSK9 antibody and the antigen-binding fragment thereof in preparing 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, then transiently transfecting CHO3E7 cells (Nanjing Jinsry Biotech Co., Ltd.), suspending and culturing for 7 days, centrifugally collecting culture liquid supernatant, and separating and purifying by nickel column affinity chromatography and molecular exclusion chromatography to obtain the recombinant human PCSK9 protein.
And (3) screening the anti-PCSK9 single-chain antibody from a 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 fully 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 using the CDR region cross-cloning method (molecular biology.2008; 46: 135-144). On the basis, the obtained high-activity anti-PCSK9 single-chain antibody is subjected to full-length amplification, and the in vitro and in vivo biological activity of the antibody is detected.
The antibody and the antigen binding fragment thereof obtained by the invention can be specifically bound with PCSK9, effectively inhibit the interaction between PCSK9 and LDLR, block PCSK 9-mediated degradation of LDLR, and remarkably enhance the uptake capacity of liver cells to 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 linked 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 denoted HCDR1, HCDR2, HCDR3, respectively; the complementarity determining regions CDR1, CDR2 and CDR3 of the light chain are denoted LCDR1, LCDR2, LCDR3, respectively.
More specifically, HCDR1 comprises SEQ ID NO: 1 to 6, or an amino acid sequence which is 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 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: 9-12, or any amino acid sequence shown in SEQ ID NO: 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 any amino acid sequence as set forth in SEQ ID NO: 13-15, 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 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: 17-24, or any amino acid sequence as set forth in SEQ ID NO: 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 any amino acid sequence as set forth in SEQ ID NO: 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: 36-46, or any amino acid sequence as set forth in SEQ ID NO: 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 comprises 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 said 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 comprising a fluorescent substance, a luminescent substance, a colored substance, a radioisotope or an enzyme.
Use of the anti-PCSK9 antibody or antigen-binding fragment thereof in the preparation of a reagent for detecting the presence of PCSK9 in a sample a test sample 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 indicates the presence of PCSK 9.
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 includes hypercholesterolemia, statin intolerance, hyperlipidemia, non-alcoholic fatty liver disease, obesity, diabetes, atherosclerosis, cardiovascular and cerebrovascular diseases, nephropathy, and the hypercholesterolemia includes 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 metabolism diseases.
Has the advantages that:
(1) the invention adopts a fully human phage antibody library to screen the antibodies targeting PCSK9, the obtained antibodies are fully human, the immunogenicity is reduced to the greatest extent, the human anti-mouse immune response is eliminated, and the half-life of the antibody drug is prolonged (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-.
(3) The full-length antibody Fc segment is modified, so that the affinity to an Fc gamma receptor 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; PCSK 9: PCSK 9-encoding gene; CMV: a CMV promoter; 6 × His tag: a histidine tag.
FIG. 2: purified recombinant human PCSK9 protein was identified by 10% (w/v) SDS-PAGE and Western Blot analysis.
sds-PAGE analysis to identify purified recombinant human PCSK9 protein, M: a protein Marker; lane 1: purified recombinant human PCSK9 protein. Western Blot to identify purified recombinant human PCSK9 protein.
FIG. 3: phage ELISA detects the relative affinity of the screened anti-human PCSK9 single-chain antibody positive clone and human PCSK9 protein. Wherein, PCSK 9: coating 5 mug/mL human PCSK 9; BSA: coating with 5 mug/mL BSA; M13K 07: helper phage M13K07 not displaying any single chain antibodies; n-3, means ± SEM.
FIG. 4: pET27b-AP2 plasmid map. Kan: a kanamycin resistance gene; AP 2: a
FIG. 5: 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 eluent containing 500mM imidazole.
Western Blot identifies the single-
FIG. 6: CDR region amino acid conservation and alternative sequence (preference) analysis of the parent single
FIG. 7: phage ELISA detects the relative affinity of the CDR region mutant (AP2M No. 1-16) of the single-chain antibody AP2 and the human PCSK9 antigen protein. Wherein, PCSK 9: 5 mug/mL human PCSK9 coating; BSA: coating with 5 mu g/mL BSA; M13K 07: 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 (AP2M No. 1-16) thereof. 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 demarcated 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 (AP2M No. 1-16) of the purified single-chain antibody AP2 were identified by 12% (w/v) SDS-PAGE.
A.M: a protein Marker; lanes 1-8: CDR region mutants of AP2 (AP2M Nos. 1-8).
B.M: a protein Marker; lanes 9-16: CDR region mutants of AP2 (AP2M No. 9-16); 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 (AP2M Nos. 1-16) on the LDLR protein level and LDL-C uptake of HepG2 cells.
A. Inhibition of PCSK 9-mediated cell surface LDLR degradation of HepG2, wherein P<0.05vs PCSK9group；#P<0.05vs AP2 group；n＝3,means±SEM。
B. Inhibition of PCSK 9-mediated down-regulation of LDL-C uptake by HepG2 cells, wherein P<0.05vs PCSK9group；#P<0.05vs AP2 group；n＝3,means±SEM。
FIG. 11: heavy chain variable region, light chain variable region and CDR region sequences of a single-chain antibody AP2 and cross cloning mutants (AP2M No. 17-25) thereof. 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 demarcated 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 (AP2MNo.17-25) were identified by 12% (w/v) SDS-PAGE. M: a protein Marker; lanes 1-9: cross-cloned mutants of the single-chain antibody AP2 (AP2M Nos. 17-25).
FIG. 13: effect of cross-cloned mutants of the single chain antibody AP2 (AP2M Nos. 17-25) on the LDLR protein level and LDL-C uptake level of HepG2 cells.
A. Inhibition of human PCSK 9-mediated degradation of HepG2 cell surface LDLR, wherein: p<0.05vs PCSK9group；#P<0.05vs AP2 group；n＝3,means±SEM。
B. Inhibition of human PCSK 9-mediated down-regulation of LDL-C uptake by HepG2 cells, wherein: p<0.05vsPCSK9 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 mutant (AP2M No.18, 21) on LDLR protein level in liver cells of mice in hyperlipidemia model after administration. Wherein: p<0.05vs Model group；**P<0.01vs Model group；#P<0.05vs AP2 group；##P<0.01vs AP2group；ΔΔP<0.01vsNormalgroup；n＝3,means±SEM。
FIG. 15: pTT5-AP2/AP2M-HC and pTT5-AP2/AP2M-LC recombinant expression plasmid maps. Amp: an ampicillin resistance gene; AP2/AP 2M-HC: an AP2 or AP2M heavy chain encoding gene; AP2/AP 2M-LC: an AP2 or AP2M light chain encoding gene; CMV: the CMV promoter.
FIG. 16: the purified full-length anti-human PCSK9 antibody (note: FAP 2: full-length antibody of parental single-
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: FAP 2: full-length antibody of parent single-
A. Inhibition of human PCSK 9-mediated degradation of HepG2 cell surface LDLR, wherein: p<0.05vs PCSK9group；**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 full-length anti-human PCSK9 antibody on the level of LDLR protein in hepatocytes of a hyperlipemia model mouse after caudal vein administration (note: FAP2M 21: full-length antibody which is a single-chain antibody AP2 cross-cloned mutant (AP2M No. 21); Alirocumab: positive control full-length antibody). Wherein: p<0.05vs Model group；**P<0.01vsModel 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 present invention is further illustrated by the following specific 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.