WO2021159235A1 - Pcsk9的单域抗体及其应用 - Google Patents

Pcsk9的单域抗体及其应用 Download PDF

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WO2021159235A1
WO2021159235A1 PCT/CN2020/074624 CN2020074624W WO2021159235A1 WO 2021159235 A1 WO2021159235 A1 WO 2021159235A1 CN 2020074624 W CN2020074624 W CN 2020074624W WO 2021159235 A1 WO2021159235 A1 WO 2021159235A1
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Prior art keywords
antibody
seq
pcsk9
antigen
sequence
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PCT/CN2020/074624
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English (en)
French (fr)
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杨乃波
高小艳
李新洋
张新华
王媚娘
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深圳华大生命科学研究院
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Priority to PCT/CN2020/074624 priority Critical patent/WO2021159235A1/zh
Priority to CN202080095724.2A priority patent/CN115052899B/zh
Publication of WO2021159235A1 publication Critical patent/WO2021159235A1/zh
Priority to US17/818,631 priority patent/US20230091895A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to the field of biotechnology. Specifically, the present invention relates to antibodies to PCSK9 and their applications. More specifically, the present invention relates to antibodies or antigen-binding fragments thereof, nucleic acid molecules, expression vectors, recombinant cells, and drugs capable of specifically recognizing PCSK9. Composition, pharmaceutical use and kit for detecting TrkA.
  • Cardiovascular disease is a serious threat to human beings and is the leading cause of death for all population groups.
  • the drugs used to lower cholesterol on the market mainly include statins, cholesterol absorption inhibitors, and probucol.
  • statins have performed well in the treatment of cardiovascular diseases, with their widespread use, possible drawbacks have gradually been discovered.
  • statins have a variety of side effects, such as abnormal blood sugar, muscle toxicity, memory and cognitive impairment, etc.
  • the incidence of side effects is as high as 20%. Severe side effects can lead to rhabdomyolysis and acute renal failure. A considerable number of patients stop treatment because they cannot tolerate the muscle pain caused by the side effects.
  • PCSK9 Proprotein convertase subtilisin/kexin type 9
  • PCSK9 a new proprotein convertase of the subfamily subfamily
  • LDL-R low-density lipoprotein receptor
  • LDL-C density lipoprotein cholesterol
  • PCSK9 gain-of-function mutations
  • loss-of-function mutations there are two types of mutations in PCSK9, gain-of-function mutations and loss-of-function mutations. Ethnic trials have shown that several PCSK9 “function-gaining” mutations often occur in individuals with autosomal dominant hypercholesterolemia, while PCSK9 “loss-of-function” mutations are associated with a decrease in plasma cholesterol. Individuals with PCSK9 loss-of-function mutations suffer from coronary heart disease. The risk is significantly reduced. In 2005, Hobbs et al. reported on Dallas Heart Study that the LDL-C level in individuals carrying the PCSK9 nonsense mutation gene would be 28% lower than that of the average person; in 2006, Hobbs et al. published the effect of PCSK9 gene mutation on coronary heart disease.
  • PCSK9 inhibitors can also inhibit the NF- ⁇ B channel, thereby reducing the risk of acute coronary syndromes such as thrombosis, inflammation, and activation of vascular endothelial cells.
  • potential research projects in the field of PCSK9 inhibitors include inhibitory protein antibodies, siRNA, anti-sense oligonucleotides, and small molecule inhibitors.
  • Monoclonal antibody drugs are currently the main field of PCSK9 inhibitor research due to their strong targeting, high specificity and low side effects. Studies at the animal level showed that after adding neutralizing anti-PCSK9 antibodies, the expression level of LDL-R in the liver of mice increased significantly, and the concentration of LDL-C in the blood decreased by 30%.
  • the PCSK9 monoclonal antibody also shows a significant effect, and the effect of reducing LDL-C levels can be maintained for more than several weeks. So far, no anti-PCSK9 protein monoclonal antibodies have been found to have obvious side effects. Only reports have reported mild side effects such as local injection reactions, diarrhea and headaches. Sanofi's Praluent (Alirocumab) and Amgen's Repatha (evolocumab) are the only two humanized PCSK9 antibodies (whole IgG) approved in the global market. According to Thomson Reuters' ranking of potential sales of approved drugs in 2015, by 2019, the sales scale of the drug will reach 4.414 billion U.S. dollars, and the sales scale of the latter will reach 1.862 billion U.S. dollars.
  • Antibody drugs are currently the main direction of new drug research and development, and have been widely used in the diagnosis, prevention and treatment of infectious diseases, and biological science research. As of 2015, 48 antibody drugs have been successfully marketed, and 7 antibody drugs have been successfully approved between April 2014 and March 2015 alone. In 2015, 6 of the top 10 drugs sold globally were antibody drugs. Since Hamers et al. discovered heavy chain antibodies that naturally lack light chains in camel blood in 1993, single-domain antibodies (Nanobody, Nb) have gradually replaced other small antibodies, and have gradually become a hot spot in the development of new antibody drugs. Nb is usually only about 15KDa, which is about one-tenth the size of traditional antibodies. There are disulfide bonds inside and a large number of hydrophilic residues on the surface.
  • Nb lacks the Fc segment and light chain.
  • the nature makes it able to recognize hidden epitopes or small epitopes that traditional antibodies cannot recognize, and avoid complement reactions; in addition, single-domain antibodies also have high stability, low toxicity, strong solubility, easy target screening, and easy in prokaryotic Direct expression in microorganisms, good economics and many other advantages.
  • Sequence homology analysis showed that the VHH germline gene sequence of camel Nb is highly homologous to human VH3, but CDR1 and CDR3 are slightly longer than humans, and CDR3 protrudes outward in the tertiary structure, so it is inferred that it has higher antigen binding. The specificity and affinity.
  • Nb is gradually being developed as a monoclonal antibody drug for disease diagnosis and treatment, and is widely used in the development of enzyme inhibitors, tumors, infections and inflammations and other biological inhibitors.
  • small size of a single domain antibody provides many advantages for its therapeutic function, small molecule proteins are extremely easy to be eliminated in the body.
  • Transforming Nb into target enzymes, transmembrane proteins, or bivalentization through genetic engineering can effectively improve antibody activity and stability to achieve research purposes.
  • the effectiveness of bivalent single-domain antibodies is at least 60 times that of monovalent single-domain antibodies, and has a longer action time in animals, effectively delaying the death of animals.
  • the prospect of antibody drugs is huge, but domestic antibody drugs are still in their infancy.
  • PCSK9 antibodies The existing technology for the development of PCSK9 antibodies is focused on traditional mouse-derived antibodies, but it is difficult to express large amounts of traditional antibodies or to humanize antibodies, which is time-consuming, expensive, and the availability of effective antibodies is low, which severely limits PCSK9.
  • antibody inhibitors especially domestic antibody drugs, is just in its infancy and cannot meet the needs of patients with cerebrovascular disease (CVD).
  • the inventor of the present application immunized camels with PCSK9 antigen, collected peripheral blood cells (PBMC) of the immunized camels, separated PCSK9 affinity lymphocytes, extracted total RNA, and cloned the V region of camel heavy chain antibodies using Nest-PCR technology. Insert it into a phage plasmid to construct a phage expression library, and then perform multiple rounds of screening of PCSK9 antigen by phage display technology. Finally, the high-affinity antibodies obtained from the screening are expressed in prokaryotic cells and purified by enzyme-linked immunosorbent assay. (ELISA) and surface plasmon resonance (SPR) to verify the affinity and binding constant of the obtained single domain antibody. The PCSK9 single domain antibody with high affinity was successfully screened.
  • PBMC peripheral blood cells
  • SPR surface plasmon resonance
  • the present invention provides an antibody or antigen-binding fragment thereof capable of specifically recognizing PCSK9.
  • the antibody includes the heavy chain variable region CDR1 shown in SEQ ID NO: 1, 2 and 3 or an amino acid sequence having at least 95% identity with SEQ ID NO: 1, 2 and 3 , CDR2, CDR3 sequence.
  • IYIGGGST SEQ ID NO: 2.
  • the heavy chain variable region CDR1 has the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence that is at least 95% identical to SEQ ID NO:1;
  • the heavy chain variable region CDR2 has the amino acid sequence shown in SEQ ID NO: 2
  • the heavy chain variable region CDR3 has the amino acid sequence shown in SEQ ID NO: 3 or the amino acid sequence with SEQ ID NO: 3 at least 95% identity .
  • the antibodies according to the embodiments of the present invention can specifically bind to PCSK9 and inhibit the activity of PCSK9.
  • the aforementioned antibody may further include at least one of the following additional technical features:
  • the antibody contains a heavy chain framework region sequence, and at least a part of the heavy chain framework region sequence is derived from at least one of a camel-derived antibody, a murine antibody, a human antibody, a primate-derived antibody or a mutant thereof. one.
  • the heavy chain framework region sequence is derived from a camel-derived antibody.
  • the heavy chain framework region sequence has an amino acid sequence shown in any one of SEQ ID NO: 4-15.
  • the heavy chain framework region sequence has an amino acid sequence shown in any one of SEQ ID NO: 8-15.
  • the inventors made certain amino acid mutations in the heavy chain framework region of the amino acid sequence shown in SEQ ID NO: 4 to 7, and obtained the heavy chain framework region of the amino acid sequence shown in SEQ ID NO: 8 to 15.
  • the inventor found that the heavy chain framework region of the amino acid sequence shown in SEQ ID NO: 8 to 15 can significantly increase the degree of humanization of the antibody and reduce the immunogenicity of the antibody.
  • the antibody has a heavy chain variable region with an amino acid sequence as shown in any one of SEQ ID NO: 16-25.
  • the antibody has a heavy chain variable region with an amino acid sequence as shown in any one of SEQ ID NO: 17-25.
  • the antibody contains a heavy chain constant region, and at least a part of at least one of the heavy chain constant regions is derived from at least one of a murine antibody, a human antibody, a primate antibody or a mutant thereof .
  • the half-life of the antibody in vivo is further increased, and the stability of the antibody is improved.
  • the heavy chain constant region of the antibody is derived from a human IgG antibody or a mutant thereof.
  • the heavy chain constant region of the antibody is derived from human IgG4.
  • the full-length sequence of the antibody constant region is shown in SEQ ID NO: 26.
  • PPCPSCP is a hinge region sequence, APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK CH2 constant region, GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK CH3 constant region.
  • the antibody has a heavy chain with an amino acid sequence shown in any one of SEQ ID NO: 27 to 36.
  • the above-mentioned antibody with the heavy chain of the amino acid sequence shown in any one of SEQ ID NO: 27 to 36 has high affinity for PCSK9, and has a prolonged half-life in vivo and higher stability.
  • the antibody is a small molecule antibody.
  • the small molecule antibody includes at least one of a single domain antibody, a Fab antibody, an Fv antibody, and a minimum recognition unit.
  • the antibody is a single domain antibody.
  • the antibody according to the embodiment of the present invention has strong resistance to heat and PH, higher affinity with the antigen PCSK9, high stability, low toxicity, strong solubility and easy direct expression in prokaryotic microorganisms , Good economy and many other advantages.
  • the present invention proposes a nucleic acid molecule.
  • the nucleic acid molecule encodes any of the aforementioned antibodies or antigen-binding fragments thereof.
  • the aforementioned nucleic acid molecule may further include at least one of the following additional technical features:
  • the nucleic acid molecule is DNA.
  • the nucleic acid molecule has a nucleotide sequence as shown in any one of SEQ ID NO: 37-46.
  • nucleotide sequence shown in SEQ ID NO: 37 encodes the heavy chain variable region of SEQ ID NO: 16
  • the nucleotide sequence shown in SEQ ID NO: 38 encodes the heavy chain variable region of SEQ ID NO: 17
  • the nucleotide sequence shown in ID NO: 39 encodes the heavy chain variable region of SEQ ID NO: 18, and the nucleotide sequence shown in SEQ ID NO: 40 encodes the heavy chain variable region of SEQ ID NO: 19, SEQ ID NO :
  • the nucleotide sequence shown in 41 encodes the heavy chain variable region of SEQ ID NO: 20
  • the nucleotide sequence shown in SEQ ID NO: 42 encodes the heavy chain variable region of SEQ ID NO: 21, SEQ ID NO:
  • the nucleotide sequence shown in 43 encodes the heavy chain variable region of SEQ ID NO: 22, and the nucleotide sequence shown in SEQ ID NO: 44 encodes the heavy chain variable region of SEQ ID NO: 23, which is shown in SEQ ID NO: 45.
  • the present invention proposes an expression vector.
  • the expression vector carries the aforementioned nucleic acid molecule.
  • the aforementioned antibody is expressed under conditions suitable for protein expression, thereby obtaining an antibody with high PCSK9 affinity activity.
  • the above-mentioned expression vector may further include at least one of the following additional technical features:
  • the expression vector is a prokaryotic expression vector.
  • the antibody according to the embodiment of the present invention is easier to express in the prokaryotic system, and then selecting a suitable prokaryotic expression vector can further improve the expression of the antibody in the prokaryotic system.
  • the present invention proposes a recombinant cell.
  • the recombinant cell carries the aforementioned nucleic acid molecule, or expresses the aforementioned antibody or antigen-binding fragment thereof.
  • the aforementioned recombinant cell may further include at least one of the following additional technical features:
  • the recombinant cell is obtained by introducing the aforementioned expression vector into a host cell.
  • the expression vector is introduced into the host cell by a method of electrotransduction.
  • the recombinant cell is a prokaryotic cell.
  • the present invention proposes a pharmaceutical composition.
  • the pharmaceutical composition contains the aforementioned antibody, the aforementioned nucleic acid molecule, the aforementioned expression vector or the aforementioned recombinant cell.
  • the pharmaceutical composition according to the embodiment of the present invention can specifically inhibit PCSK9, and is used to effectively treat or prevent PCSK9-related diseases with small side effects and long drug effect time.
  • the present invention proposes that the aforementioned antibody, the aforementioned nucleic acid molecule, the aforementioned expression vector or the aforementioned recombinant cell, and the aforementioned pharmaceutical composition are used in the preparation of medicines.
  • the medicine is used to treat or prevent PCSK9-related diseases.
  • the medicine is used to treat or prevent PCSK9-related diseases, and has the advantages of small side effects and long pharmacological effects.
  • the above-mentioned use may further include at least one of the following additional technical features:
  • the drug is used to treat or prevent hyperlipidemia, hypercholesterolemia, and/or diseases caused by atherosclerosis, preferably cardiovascular disease in mammals, preferably in humans, Stroke or peripheral vascular disease.
  • the present invention provides a kit for detecting PCSK9.
  • the kit includes the aforementioned antibody.
  • the kit according to the embodiment of the present invention can be used for the specific detection of PCSK9
  • the present invention proposes the use of the aforementioned antibody, nucleic acid molecule, expression vector or recombinant cell in the preparation of a kit for detecting PCSK9 or diagnosing PCSK9-related diseases.
  • Figure 1 is a gel electrophoresis identification diagram of a VHH fragment of a camel heavy chain antibody amplified by Nest-PCR according to an embodiment of the present invention
  • FIG. 2 is a gel electrophoresis result diagram of the recombination efficiency of the VHH fragments in the phage display library constructed according to the PCR verification of the embodiment of the present invention
  • Figure 3 is a diagram showing the screening results of the elutriated PCSK9 single domain antibody monoclonal ELISA according to an embodiment of the present invention
  • Figure 4 is a diagram showing the results of monoclonal PCR screening of elutriated PCSK9 single domain antibodies according to an embodiment of the present invention
  • Figure 5 is a diagram showing the results of SDS-PAGE verification of the expression and purification of the PCSK9 single domain antibody according to an embodiment of the present invention
  • Figure 6 is a graph showing the results of ELISA verification of the affinity of purified PCSK9 single domain antibodies according to an embodiment of the present invention.
  • Fig. 7 is a graph showing the results of detecting VHH4 affinity by SPR according to an embodiment of the present invention.
  • Fig. 8 is a graph showing the results of detecting the affinity of VHH-Z3 by SPR according to an embodiment of the present invention.
  • Figure 9 is a graph of the results of SPR detecting VHH-Z3-Fc affinity according to an embodiment of the present invention.
  • Figure 10 is a graph showing the results of in vitro pharmacodynamic verification of the PCSK9 antibody according to an embodiment of the present invention.
  • Figure 11 is a graph showing the results of in vivo pharmacodynamic verification of the PCSK9 antibody according to an embodiment of the present invention.
  • single domain antibody is a heavy chain antibody found in camel blood that naturally lacks the light chain, and only includes the heavy chain H chain with a heavier molecular weight.
  • the amino-terminal (N-terminal) amino acid sequence of the peptide chain has great changes, called the variable region (V region), and the carboxyl terminal (C-terminal) is relatively stable with little change, and is called the constant region (C region).
  • the V region of the H chain is called VH.
  • the amino acid composition and arrangement order of certain regions have a higher degree of variation, which is called hypervariable region (Hypervariable region, HVR).
  • the hypervariable region is the position where antigen and antibody bind, so it is also called determinant complementation.
  • Complementarity-determining region CDR
  • the variable region of the heavy chain has three CDR regions. CDR1 and CDR3 are slightly longer than humans, and CDR3 protrudes outward in the tertiary structure. Therefore, it is speculated that single-domain antibodies have higher antigen binding specificity and affinity than traditional antibodies.
  • the present invention uses the PCSK9 antigen expressed by CHO cells to immunize camels, and obtains high specificity and high affinity anti-PCSK9 single domain antibody (Nanobody, Nb).
  • the antibody can specifically bind to the PCSK9 antigen, so that hyperlipidemia and cardiovascular and cerebrovascular diseases can be treated or prevented in a targeted manner.
  • the present invention provides an antibody or antigen-binding fragment capable of specifically recognizing PCSK9, said antibody containing a CDR sequence selected from at least one of the following or an amino acid sequence having at least 95% identity with it: Chain variable region CDR sequence: SEQ ID NO: 1 ⁇ 3.
  • the antibodies or antigen-binding fragments provided by the present invention have conservative amino acid substitutions compared to the heavy chains described above.
  • Antigen-binding fragment refers to an antibody fragment that retains the ability to specifically bind to an antigen (PCSK9).
  • Constant amino acid substitution refers to the substitution of an amino acid by another amino acid that is biologically, chemically or structurally similar.
  • Biologically similar means that the substitution does not destroy the biological activity of the PCSK9 antibody or the PCSK9 antigen.
  • Structurally similar means that amino acids have side chains of similar length, such as alanine, glycine, or serine, or side chains of similar size.
  • Chemical similarity means that the amino acids have the same charge or are both hydrophilic or hydrophobic. For example, the hydrophobic residues isoleucine, valine, leucine or methionine are substituted for each other.
  • use polar amino acids such as arginine for lysine, glutamic acid for aspartic acid, glutamine for asparagine, serine for threonine and so on.
  • the present invention provides an antibody or antigen-binding fragment having a heavy chain variable region with an amino acid sequence shown in any one of SEQ ID NO: 16-25.
  • the inventors can obtain the CDR regions of the above-mentioned anti-heavy chain variable region sequence (as shown in SEQ ID NO: 1 to 3) through the antibody sequence alignment database (NCBI, IMGT).
  • the heavy chain variable region sequence of the antibody or antigen-binding fragment has conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 16-25. Of course, these conservative amino acid substitutions will not change the biological function of the antibody or antigen-binding fragment. In some specific ways, these conservative amino acid substitutions can occur on amino acids other than the CDR region in the variable region of the heavy chain.
  • the present invention provides an anti-PCSK9 antibody having a heavy chain with the amino acid sequence shown in any one of SEQ ID NO: 27 to 36.
  • the present invention provides an anti-PCSK9 single domain antibody, which has a heavy chain variable region of the amino acid sequence shown in any one of SEQ ID NO: 17-25 or any of SEQ ID NO: 28-36. A heavy chain of the amino acid sequence shown.
  • nucleic acid molecules expressing these antibodies can be connected to different vectors and then expressed in different cells to obtain corresponding antibodies.
  • the present invention also provides an isolated nucleic acid molecule that encodes the aforementioned antibody or antigen-binding fragment.
  • the isolated nucleic acid molecule has a nucleotide sequence as shown in any one of SEQ ID NO: 37-46.
  • the isolated nucleic acid molecule has at least 90% or more homology with the nucleotide sequence shown in SEQ ID NO: 37 to 46, preferably more than 95% homology, and more preferably It has 98%, 99% or more homology.
  • These sequences that have homology with the nucleotide sequence shown in SEQ ID NO: 37 to 46 can express amino acid sequences similar to SEQ ID NO: 16 to 25, so that they can specifically bind to the PCSK9 antigen to achieve antibody targets Directional function.
  • the present invention also provides an expression vector comprising the above-mentioned isolated nucleic acid molecule.
  • the isolated polynucleotide When the isolated polynucleotide is connected to the vector, the polynucleotide can be directly or indirectly connected to the control elements on the vector, as long as these control elements can control the translation and expression of the polynucleotide.
  • these control elements can come directly from the carrier itself, or they can be exogenous, that is, they do not come from the carrier itself.
  • the polynucleotide can be operably linked to the control element.
  • operably linked refers to the connection of the foreign gene to the vector, so that the control elements in the vector, such as transcription control sequence and translation control sequence, etc., can play their intended role in regulating the transcription and translation of the foreign gene. Function.
  • the polynucleotides used to encode the antibody heavy chain can be inserted into different vectors independently, and it is common to insert into the same vector.
  • Commonly used vectors can be, for example, plasmids, bacteriophages, and the like. For example, Plasmid-X plasmid.
  • the present invention also provides a recombinant cell, which contains the expression vector.
  • the expression vector can be introduced into prokaryotic cells to construct recombinant cells, and then these recombinant cells can be used to express the antibodies or antigen-binding fragments provided by the present invention. By culturing the recombinant cells, the corresponding antibodies can be obtained.
  • the pharmaceutical composition The pharmaceutical composition, the kit, the pharmaceutical use and the use in the preparation of the kit.
  • the present invention also provides a pharmaceutical composition, which comprises the aforementioned antibody or antigen-binding fragment and a pharmaceutically acceptable carrier.
  • the anti-PCSK9 antibodies provided herein can be incorporated into pharmaceutical compositions suitable for administration to a subject.
  • these pharmaceutical compositions include the anti-PCSK9 antibody provided herein and a pharmaceutically acceptable carrier.
  • the "pharmaceutically acceptable carrier” may include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Specific examples may be one or more of water, saline, phosphate buffered saline, glucose, glycerol, ethanol, etc., and combinations thereof.
  • the pharmaceutical composition includes isotonic agents, such as sugars, polyalcohols (such as mannitol, sorbitol), or sodium chloride.
  • isotonic agents such as sugars, polyalcohols (such as mannitol, sorbitol), or sodium chloride.
  • the pharmaceutically acceptable carrier may also include minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, to extend the shelf life or efficacy of the antibody.
  • the antibodies of the present invention can be incorporated into pharmaceutical compositions suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular).
  • parenteral administration e.g., intravenous, subcutaneous, intraperitoneal, intramuscular
  • These pharmaceutical compositions can be prepared in various forms.
  • liquid, semi-solid and solid dosage forms including but not limited to liquid solutions (for example, injection solutions and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories.
  • Typical pharmaceutical compositions are in the form of injection solutions or infusion solutions.
  • the antibody can be administered by intravenous infusion or injection, or intramuscular or subcutaneous injection.
  • the anti-PCSK9 antibody herein can also be made into a part of a kit or other diagnostic reagents as needed.
  • the present invention also provides a kit, which includes the aforementioned PCSK9 antibody.
  • the kit provided by the present invention for example, it can be used for immunoblotting, immunoprecipitation, etc., which involve the use of PCSK9 antigen and antibody specific binding properties for detection.
  • kits may contain any one or more of the following: antagonists, anti-PCSK9 antibodies or drug reference materials; protein purification columns; immunoglobulin affinity purification buffers; cell assay diluents; instructions or literature, etc.
  • Anti-PCSK9 antibodies can be used in different types of diagnostic tests, for example, it can detect the presence of various diseases or drugs, toxins or other proteins in vitro or in vivo.
  • the subject's serum or blood can be tested for related diseases.
  • related diseases may include PCSK9 related diseases, such as lipemia, hypercholesterolemia, and/or diseases caused by atherosclerosis, cardiovascular disease, stroke or peripheral vascular disease and so on.
  • the antibodies provided herein can also be used for radioimmunoassay and radioimmunotherapy of the above-mentioned diseases.
  • the anti-PCSK9 antibody provided by the present invention can be provided to the subject.
  • the present invention provides a method for treating the above-mentioned diseases, which comprises administering the antibody or antigen-binding fragment thereof provided by the present invention to a subject in need.
  • the present invention uses the PCSK9 antigen expressed in eukaryotic cells to immunize camels, and obtains a high-quality PCSK9 immune single domain antibody antibody library through flow sorting of affinity lymphocytes. Coat the PCSK9 antigen on the enzyme-labeled plate, screen the PCSK9 immune single domain antibody library using phage display technology, and then transform the screened single domain antibody into the E. coli expression system for large-scale expression, so that it can be expressed in a relatively short time A monoclonal single-domain antibody strain of PCSK9 with high affinity is obtained here.
  • the inventors independently designed nine antibodies of VHH-Hu and VHH-Z1-8 based on the results of phage screening.
  • Lymphocytes are separated from blood samples collected before and after each immunization.
  • the separation method is as follows:
  • cryopreserved lymphocytes Take a portion of cryopreserved lymphocytes, add 1mL Trizol, and after standing at room temperature for 10 minutes, add 0.2mL of chloroform, shake vigorously, and stand at room temperature. After the solution is separated (about 10 minutes), centrifuge at 12,000 rpm, and then collect the upper aqueous phase.
  • RNA precipitation was dissolved in nuclease-free water, and 1 ⁇ L was used for concentration and purity determination.
  • RNA Take 1 ⁇ g RNA, use SuperScript TM III First-Strand Synthesis SuperMix (Invitrogen) kit for cDNA synthesis, use Oligo dT as reverse transcription primer, and freeze the synthesized cDNA at -20°C;
  • the PCR reaction conditions are as follows:
  • Reaction conditions 95°C, 5min; 94°C, 1min; 57°C, 1min; 72°C, 1min per cycle; 72°C, 7min; amplification 35 cycles.
  • Reaction conditions 95°C, 5min; 94°C, 45'; 60°C, 45'; 72°C, 45' per cycle; 72°C, 7min; 25 cycles of amplification.
  • Figure 1 shows the results of nest-PCR electrophoresis.
  • the target gene fragment of the first round of PCR is at 700bp.
  • the gel is cut to recover the target band, and the second is In the round of PCR, the target gene fragment is at 500bp, and the gel is cut to recover the target band, that is, the VHH fragment.
  • PCR Purification Kit QIAGEN
  • helper phage to amplify and rescue the phage library of PCSK9 single domain antibody.
  • PCSK9 antigen Take 100ng of PCSK9 antigen to coat the ELISA plate, incubate overnight at 4°C. The next day, add the rescued PCSK9 single domain antibody phage and incubate at room temperature for 2 hours; wash the wells with PBST 10 times, add 100 ⁇ L of triethylamine, and incubate at room temperature for 30 min.
  • the collected phage are the PCSK9 single domain antibody phage obtained by affinity panning. Library; take 10 ⁇ L of infected TG cells to spread on the plate for the determination of the number of clones after screening, and the remaining phage after screening for amplification.
  • the amplification and rescue methods are the same as those in Example 1(7).
  • the obtained PBS suspension which is the amplified phage after the first round of screening, is stored at 4°C and used for the next round of screening; according to the same screening steps as above, Decrease the amount of antigen successively, and screen 3-4 rounds.
  • the ELISA plate was coated with 100ng of PCSK9 antigen at 4°C overnight; the next day was blocked with 2% BSA at room temperature for 1 hour; the experimental group was added with the phage amplified after each round of panning, and the control group was added with the same amount of wild-type phage, at room temperature , Incubate for 2h; wash with PBST 10 times to remove unbound phages; add HRP-labeled anti-M13 antibody and incubate for 1h at room temperature; add chromogenic solution and react for 10-30min in the dark, and measure the absorbance value.
  • the absorbance value follows the elutriation The frequency gradually increased and stabilized from the third to the fourth round of panning, indicating that the specific antibodies were enriched.
  • the ELISA plate was coated with 100ng of PCSK9 antigen and incubated overnight at 4°C; take the phage-coated plate obtained in the last round of screening, randomly pick 38 single clones in 1mL medium, culture it to logarithmic phase at 37°C, and add 1mM IPTG was induced overnight; the next day, centrifuged to collect the bacterial pellet, after breaking, centrifuged at 5,000g for 15min to collect the supernatant; at the same time, the ELISA plate was taken, and 2% BSA was added to block at room temperature for 1 hour; each well of the experimental group was added with monoclonal broken supernatant.
  • the DNA of the 30 positive clones obtained in (4) was extracted to perform PCR verification on the inserted fragments, as shown in Figure 4, the clones that were verified as positive by PCR were subjected to sequencing analysis.
  • the sequencing results showed that two nucleotide sequences were obtained, and their amino acid sequences were analyzed.
  • One of the sequences has the structure of a typical single-domain antibody, which is composed of framework regions (FR1, FR2, FR3 and FR4) and complementarity determining regions ( CDR1, CDR2 and CDR3) constitute.
  • amino acid sequence of this single domain antibody VHH4 monoclonal is as follows:
  • FR1 is QVQLQESGGGSVQAGGSLRLSCTVS (SEQ ID NO: 4)
  • FR2 is MGWFRQAPGKEHEGVAS (SEQ ID NO: 5)
  • FR3 is YYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYC (SEQ ID: SEQ ID NO: 6)
  • FR4 is WDNC (SEQ ID: SEQ ID NO: 6) NO:1)
  • the sequence of CDR 2 is IYIGGGST (SEQ ID NO: 2)
  • the sequence of CDR 3 is AVGCQGLVDFGY (SEQ ID NO: 3).
  • VHH-Hu and VHHZ1-8 In order to increase the degree of humanization of single domain antibodies and reduce immunogenicity, certain amino acids in the four framework regions of VHH4 were mutated to obtain nine antibody sequences VHH-Hu and VHHZ1-8.
  • PCSK9 antibody protein VHH-Hu its amino acid sequence is: EVQLLESGGGLVQPGGSLRLSCAASGYTYSSNCMGWVRQAPGKGLEWVSSIYIGGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVGCQGLVDFGYWDQGTQVTVSS (SEQ ID NO: 17).
  • framework region 1 is EVQLLESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 8),
  • the sequence of framework region 2 is MGWVRQAPGKGLEWVSS (SEQ ID NO: 9),
  • the sequence of framework region 3 is YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO: 10),
  • the sequence of framework region 4 is WDQGTQVTVSS (SEQ ID NO: 7),
  • CDR1 The sequence of CDR1 is GYTYSSNC (SEQ ID NO:1),
  • CDR2 is IYIGGGST (SEQ ID NO: 2)
  • the sequence of CDR3 is AVGCQGLVDFGY (SEQ ID NO: 3).
  • PCSK9 antibody protein VHH-Z1 its amino acid sequence is:
  • framework region 1 is EVQLLESGGGLVQPGGSLRLSCTVS (SEQ ID NO: 11),
  • the sequence of framework region 2 is MGWFRQAPGKEHEGVSS (SEQ ID NO: 12),
  • the sequence of framework region 3 is YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO: 10),
  • the sequence of framework region 4 is WDQGTQVTVSS (SEQ ID NO: 7),
  • CDR1 The sequence of CDR1 is GYTYSSNC (SEQ ID NO:1),
  • CDR2 is IYIGGGST (SEQ ID NO: 2)
  • the sequence of CDR3 is AVGCQGLVDFGY (SEQ ID NO: 3).
  • PCSK9 antibody protein VHH-Z2 its amino acid sequence is:
  • framework region 1 is EVQLLESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 8),
  • the sequence of framework region 2 is MGWFRQAPGKEHEGVSS (SEQ ID NO: 12),
  • the sequence of framework region 3 is YYADSVKGRFTISQDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO: 13),
  • the sequence of framework region 4 is WDQGTQVTVSS (SEQ ID NO: 7),
  • CDR1 The sequence of CDR1 is GYTYSSNC (SEQ ID NO:1),
  • CDR2 is IYIGGGST (SEQ ID NO: 2)
  • the sequence of CDR3 is AVGCQGLVDFGY (SEQ ID NO: 3). .
  • PCSK9 antibody protein VHH-Z3 its amino acid sequence is:
  • framework region 1 is EVQLLESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 8),
  • the sequence of framework region 2 is MGWFRQAPGKEHEGVAS (SEQ ID NO: 5),
  • the sequence of framework region 3 is YYADSVKGRFTISQDNAKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO: 14),
  • the sequence of framework region 4 is WDQGTQVTVSS (SEQ ID NO: 7),
  • the sequence of CDR1 is GYTYSSNC (SEQ ID NO:1).
  • the sequence of CDR2 is IYIGGGST (SEQ ID NO: 2).
  • the sequence of CDR3 is AVGCQGLVDFGY (SEQ ID NO: 3).
  • PCSK9 antibody protein VHH-Z4 its amino acid sequence is:
  • framework region 1 is EVQLLESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 8),
  • the sequence of framework region 2 is MGWFRQAPGKEHEGVSS (SEQ ID NO: 12),
  • the sequence of framework region 3 is YYADSVKGRFTISQDNAKNTLYLQMNSLRAEDTAVYYC(seq id no:14),
  • the sequence of framework region 4 is WDQGTQVTVSS (SEQ ID NO: 7),
  • CDR1 The sequence of CDR1 is GYTYSSNC (SEQ ID NO:1),
  • CDR2 is IYIGGGST (SEQ ID NO: 2)
  • the sequence of CDR3 is AVGCQGLVDFGY (SEQ ID NO: 3).
  • PCSK9 antibody protein VHH-Z5 its amino acid sequence is:
  • framework region 1 is EVQLLESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 8),
  • the sequence of framework region 2 is MGWFRQAPGKEHEGVSS (SEQ ID NO: 12),
  • the sequence of framework region 3 is YYADSVKGRFTISQDNSKNTVYLQMNSLRAEDTAVYYC (SEQ ID NO: 15),
  • the sequence of framework region 4 is WDQGTQVTVSS (SEQ ID NO: 7),
  • CDR1 The sequence of CDR1 is GYTYSSNC (SEQ ID NO:1),
  • CDR2 is IYIGGGST (SEQ ID NO: 2)
  • the sequence of CDR3 is AVGCQGLVDFGY (SEQ ID NO: 3).
  • the PCSK9 antibody protein VHH-Z6, its amino acid sequence is:
  • framework region 1 is EVQLLESGGGLVQPGGSLRLSCTVS (SEQ ID NO: 11),
  • the sequence of framework region 2 is MGWFRQAPGKEHEGVSS (SEQ ID NO: 12),
  • the sequence of framework region 3 is YYADSVKGRFTISQDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO: 13),
  • the sequence of framework region 4 is WDQGTQVTVSS (SEQ ID NO: 7),
  • CDR1 The sequence of CDR1 is GYTYSSNC (SEQ ID NO:1),
  • CDR2 is IYIGGGST (SEQ ID NO: 2)
  • the sequence of CDR3 is AVGCQGLVDFGY (SEQ ID NO: 3).
  • the PCSK9 antibody protein VHH-Z7 its amino acid sequence is:
  • framework region 1 is EVQLLESGGGLVQPGGSLRLSCTVS (SEQ ID NO: 11),
  • the sequence of framework region 2 is MGWFRQAPGKEHEGVSS (SEQ ID NO: 12),
  • the sequence of framework region 3 is YYADSVKGRFTISQDNAKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO: 14),
  • the sequence of framework region 4 is WDQGTQVTVSS (SEQ ID NO: 7),
  • CDR1 The sequence of CDR1 is GYTYSSNC (SEQ ID NO:1),
  • CDR2 is IYIGGGST (SEQ ID NO: 2)
  • the sequence of CDR3 is AVGCQGLVDFGY (SEQ ID NO: 3).
  • PCSK9 antibody protein VHH-Z8 its amino acid sequence is:
  • framework region 1 is EVQLLESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 8),
  • the sequence of framework region 2 is MGWFRQAPGKEHEGVSS (SEQ ID NO: 12),
  • the sequence of framework region 3 is YYADSVKGRFTISQDNAKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO: 14),
  • the sequence of framework region 4 is WDQGTQVTVSS (SEQ ID NO: 7),
  • CDR1 The sequence of CDR1 is GYTYSSNC (SEQ ID NO:1),
  • CDR2 is IYIGGGST (SEQ ID NO: 2)
  • the sequence of CDR3 is AVGCQGLVDFGY (SEQ ID NO: 3).
  • the human IgG4 Fc region was fused, and nine Fc fusion antibodies of VHH-Hu-Fc and VHHZ1 ⁇ 8-Fc were obtained.
  • the sequence is shown below.
  • the sequence of the hinge region is: PPCPSCP
  • the sequence of the constant region CH2 region is:
  • the sequence of the constant region CH3 region is:
  • PCSK9 single domain antibody monoclonal transfer medium 37°C, overnight culture; the next day, use EZNA Plasmid Mini Kit I (OMEGA) extracts the plasmid, agarose gel electrophoresis and determination of the concentration, the plasmid containing the PCSK9 single domain antibody sequence is transformed into the expression bacteria HB2151, the plate is coated, and the plate is incubated overnight at 37°C.
  • OEGA EZNA Plasmid Mini Kit I
  • the PCSK9 single domain antibody was obtained by affinity purification with Ni column.
  • the Ni column was first washed with ultrapure water, and then washed with lysis solution; the crushed supernatant of the above-mentioned PCSK9 single domain antibody expression bacteria was added to the Ni column at a flow rate of 1 mL/min; with 5 column volumes of affinity solution A (20mM imidazole ) Wash away the contaminant protein, and then use an equal volume of affinity B solution (250mM imidazole) to elute the target protein, and collect the eluate; the final 15% SDS-PAGE monitors the expression and purification of the PCSK9 single domain antibody ( Figure 5 ).
  • HEK293 cells-subculture HEK293 cells with 293 serum-free CD medium (Beijing Yiqiao, item number SMM 293-TI)
  • the plasmids VHH4-Fc and VHH-Z3-Fc to be expressed and transfection reagent TF2 (Beijing Yiqiao, item number) STF02) was mixed and added to the cells, and 293 serum-free feed solution (Beijing Yiqiao, catalog number M293-SUPI-100) was added on the 1, 3, and 5 days after transfection.
  • Shaking flask culture conditions 5% CO 2 , temperature 37° C., shaker rotation speed 175 rpm.
  • the culture medium was centrifuged at 6000 rpm for 20 minutes, it was filtered with a filter, and the supernatant was taken.
  • Load and balance Load the protein A affinity chromatography column on the material solution, and rinse with the balance buffer until the baseline is stable after the sample is loaded;
  • the experimental group was coated with 100ng of PCSK9 protein, and the control group was coated with uninduced plasmid protein and BSA, and incubated overnight at 4°C; the next day, 2% BSA was added to block at room temperature for 1 hour; the purified PCSK9 antibody was added to the control.
  • Group and experimental group blank group was added PBS, room temperature, incubate 2h; PBST wash 10 times, add secondary antibody, room temperature 1h; add substrate, react for 10-20min, read the absorbance value on the microplate reader.
  • the ELISA test results ( Figure 6) show that the PCSK9 single domain antibody has a good affinity for PCSK9, and its binding activity is much higher than that of the control group.
  • the cultured Huh7 and HepG2 cells were plated in a 96-well plate (Corning, USA) at a cell density of 5 ⁇ 10 5 cells/well (resuspended in fresh DMEM containing 10% fetal bovine serum). After culturing at 37°C for 18-20h, the cells were washed with D-PBS (Gibco brand in the United States) and transferred to a serum-free culture system. A total of five groups are designed for the experiment: the first group is a blank control (without hPCSK9 and antibody). The second group is a negative control (only hPCSK9 is added, the concentration is 0.08 ⁇ M.
  • the hPCSK9 (concentration is 0.08 ⁇ M) protein preheated at 37°C and the other two groups of antibodies (VHH-Z3 and VHHZ3-Fc, the concentration is set to 0.75, respectively) ⁇ M, 0.375 ⁇ M, and 1.5 ⁇ M) were added to the above cells.
  • the fluorescence intensity can reflect the metabolic level of low-density lipoprotein LDL.
  • the results are shown in Figure 10.
  • the addition of VHH4, VHH-Z3 and VHHZ3-Fc antibodies can block the binding of PCSK9 and LDLR, thereby promoting the absorption of LDLC by LDLR on the cell surface.
  • the animal pharmacodynamic experiment was divided into seven groups, each with six rats, of which five groups were hPCSK9 transgenic mice (Tg+ mice) group and two normal control rat groups.
  • the hPCSK9 transgenic rat is realized by randomly inserting the hPCSK9 gene into the fertilized egg genome of normal rats.
  • the successfully transgenic rats were verified by PCR to verify the correct insertion of the hPCSK9 gene.
  • Tg+ rats induced hyperlipidemia symptoms after 8 weeks of high-fat diet.
  • the first four groups of transgenic rats continued to have a high-fat diet, and were injected with PBS, commercially available rebamumab (Evolocumab), VHH-Z3 and VHH-Z3-Fc into the tail vein (the injection dose was 20mg/Kg). They were named as the transgenic high-fat blank group, the transgenic high-fat positive group, the transgenic high-fat VHH-Z3 group, and the transgenic high-fat VHH-Z3-Fc group. The remaining group of transgenic rats were fed with a normal diet. Simultaneously set up two control groups of normal rats.
  • the first group was a non-GMO normal diet group
  • the second group was a non-GMO high-fat diet fed with commercially available rebamumab and named the non-GMO high-fat positive group.
  • Mice were injected on day 0 to 7 and tested on day 19.
  • the blood sample collection and total cholesterol (CHOL) and liver low-density lipoprotein levels were measured by an automatic biochemical analyzer (Bs-600, Mindray brand, China), and the results are shown in Figure 11.
  • hPCSK9 rats with hyperlipidemia can significantly reduce the levels of CHOL and LDL-C after being given positive drugs and maintain them to the end of the experiment.
  • test drug VHH-Z3-Fc also had a significant effect on reducing the levels of CHOL and LDL-C in hPCSK9 hyperlipidemia model rats, and its lipid-lowering effect was slightly better than that from day 2 to day 13 after administration. Positive drug group.
  • the test drug VHH-Z3 has no significant effect on the levels of CHOL and LDL-C in hPCSK9 hyperlipidemia model rats during the entire administration period

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Abstract

一种能够特异性识别PCSK9的抗体或其抗原结合片段。该抗体包括如SEQ ID NO:1、2和3或者与SEQ ID NO:1、2和3具有至少95％同一性的氨基酸序列所示的重链可变区CDR1、CDR2、CDR3序列。该抗体能够特异性结合PCSK9，抑制PCSK9的活性。

Description

PCSK9的单域抗体及其应用 技术领域
本发明涉及生物技术领域，具体地，本发明涉及PCSK9的抗体及其应用，更具体地，本发明涉及能够特异性识别PCSK9的抗体或其抗原结合片段、核酸分子、表达载体、重组细胞、药物组合物、制药用途以及检测TrkA的试剂盒。
背景技术
心血管疾病严重威胁着人类，是所有人口族群的首要死因。目前市面上用于降低胆固醇的药物主要有他汀类(statins)、胆固醇吸收抑制剂、普罗布考等。尽管他汀类药物心血管疾病治疗上表现出色，但随着其广泛使用，可能产生的弊端也渐渐被发现。首先，强化他汀治疗患者仍有较高心血管事件残余风险，在2年内发生的风险达22.4％；其次，有大量患者无法耐受他汀类药物，尤其是家族性高胆固醇血症患者，即使接受最大剂量的最有效的他汀类药物治疗，仍然不能达到降低LDL-C浓度的目的；最重要的是，他汀类药物存在多种副作用，如引起患者血糖异常、肌肉毒性、记忆和认知障碍等，副作用的发生率高达20％，严重的副作用会导致横纹肌溶解和急性肾功能衰竭，相当一部分患者因不能忍受副作用带来的肌肉疼痛而终止了治疗。
前蛋白转化酶枯草溶菌素9(Proprotein convertase subtilisin/kexin type 9,PCSK9)，枯草蛋白酶亚家族的一种新的前蛋白转化酶，是常染色体显性家族性高胆固醇血症的重要影响因子之一。研究发现，PCSK9除了能影响血浆胆固醇水平，调节神经细胞的凋亡，还与炎症反应有一定的相关性。目前对于PCSK9的研究主要集中在对肝脏脂质代谢的调节功能。前期的研究显示，PCSK9可通过促进肝细胞的低密度脂蛋白受体(low-density lipoprotein receptor,LDL-R)的降解，调节肝脏脂质代谢，进而影响血浆中低密度脂蛋白胆固醇(low-density lipoprotein cholesterol,LDL-C)的水平。但PCSK9存在两种突变类型，功能获得型突变和功能缺失型突变。族群试验显示，若干PCSK9“获得功能”的突变常发生于体染色体显性高胆固醇血症的个体，而PCSK9“失去功能”的突变则与血浆胆固醇减少有，PCSK9功能缺失型突变个体患冠心病的风险明显降低。2005年，Hobbs等在Dallas Heart Study上报道了携带PCSK9无义突变基因的个体中LDL-C水平会比一般人低28％；在2006，Hobbs等又发表PCSK9基因突变对冠心病的作用，该结果基于一项动脉粥样硬化风险调查，他们对9523个白人和3363个非洲裔美国人进行了长达15年的跟踪观察，发现缺失1个或2个PCSK9功能基因的人群的冠心病的发病率显著低于普通人群。Copenhagen Heart Study 发现PCSK9基因的功能性缺失会使LDL-C水平下降11-15％，冠心病患病率下降6-46％。Zimbabwe等报道了PCSK9的缺失突变可使非洲女性的LDL-C水平下降27％。PCSK9抑制剂提供了一种全新的治疗模式来对抗LDL-C，被视为他汀类之后降脂领域取得的最大进步。PCSK9抑制剂的出现，为那些服用他汀类药物时出现严重副作用的患者，及他汀类药物治疗无法达到LDL-C目标水平的患者，如遗传性高胆固醇血症患者带来了福音。
PCSK9抑制剂除了能阻止LDL-R回收，还可以抑制NF-κB通道，从而减少血栓、炎症、血管内皮细胞激活等急性冠状动脉综合症的风险。目前PCSK9抑制剂这个领域潜在的研究项目有抑制蛋白抗体、siRNA、反意寡核苷酸以及小分子抑制剂等。单克隆抗体药物因具有靶向性强、特异性高和毒副作用低等特点，是目前PCSK9抑制剂研究的主要领域。在动物水平的研究显示，加入中和抗PCSK9的抗体之后，小鼠肝脏中的LDL-R表达水平显著上升，血液中LDL-C浓度下降30％。对灵长类动物，PCSK9单克隆抗体也表现出显著的效果，LDL-C水平的下降效果可维持数周以上。到目前为止，还没有发现抗PCSK9蛋白单抗类药物有比较明显的毒副作用，只有报道称出现过局部注射反应、腹泻和头疼等较轻微的副作用。赛诺菲的Praluent(Alirocumab)和安进的Repatha(evolocumab)是目前全球市场唯一获批的两款人源化的PCSK9抗体(whole IgG)。根据汤森路透对2015年获批药物潜力销售排行榜，到2019年前者的销售规模将达到44.14亿美元，而后者的销售规模将会达到18.62亿美元。
抗体药物是目前新药研发的主要方向，在传染病的诊断、防治和生物科学研究领域都已得到了广泛的应用。到2015年截止，已经有48个抗体药物成功上市，仅2014年4月至2015年3月期间就有7个抗体药物成功获批。2015年全球销售排名前10的药物中有6个都为抗体药物。自1993年Hamers等在骆驼血液中发现了天然缺失轻链的重链抗体后，单域抗体(Nanobody,Nb)逐渐取代其他的小型抗体，逐渐成为新型抗体药物研发的热点。Nb通常只有15KDa左右，约传统抗体大小的十分之一，其内部存在二硫键，表面有大量亲水残基，对热和PH有较强的抵抗力；Nb缺乏Fc段和轻链的性质使其能够识别传统抗体无法识别的隐蔽表位或小表位，且避免了补体反应；此外，单域抗体还具有稳定性高、毒性低、可溶性强、易于靶点筛选，和易于在原核微生物中直接表达，经济性好等诸多优势。序列同源性分析显示，骆驼Nb的VHH胚系基因序列和人VH3高度同源，但CDR1和CDR3比人稍长，CDR3在三级结构中向外凸出，因而推测有更高的抗原结合的特异性和亲和力。鉴于以上优点，Nb正被逐步开发为疾病诊断和治疗方面的单抗药物，广泛的应用与酶的抑制剂，肿瘤、感染和炎症等生物抑制剂的开发上。然而，单域抗体微小的体积虽为其治疗功能提供了很多的优势，但小分子蛋白在体内极容易被消除。通过基因工程将Nb改造成靶点酶、跨膜蛋白或者双价化能够有效的提高抗体活力和稳定性等，以达到研究 目的。在对抑制病毒复制的研究中发现，双价单域抗体的有效性至少是单价单域抗体的60倍，并且在动物体内作用时间更长，有效的延迟了动物的死亡时间。抗体药物的前景巨大，但国内抗体药物仍然处于起步阶段。
因此，开发我国国产化PCSK9抗体抑制剂，满足我国国民对于抗体药物的迫切需求，具有深远而积极的意义。
发明内容
本申请是基于发明人对以下事实和问题的发现和认识作出的：
现有技术对于PCSK9抗体的开发集中在鼠源传统抗体，但传统抗体无论是大量表达，还是进行抗体人源化都比较困难，耗时长，花费高，并且有效抗体获得率低，严重限制了PCSK9抗体抑制剂的开发，特别是国内抗体药物刚刚处于起步阶段，完全不能满足脑血管疾病(CVD)患者的需求。
本申请的发明人利用PCSK9抗原免疫骆驼，采集免疫后的骆驼的外周血细胞(PBMC)，从中分离PCSK9的亲和淋巴细胞，提取总RNA，采用Nest-PCR技术克隆骆驼重链抗体的V区，将其插入到噬菌体质粒中，构建噬菌体表达文库，接着通过噬菌体展示技术对PCSK9抗原进行多轮筛选，最后将筛选获得的高亲和抗体在原核细胞进行大量表达纯化，并经酶联免疫吸附试验(ELISA)和表面等离子共振(SPR)对所获得的单域抗体的亲和力和结合常数进行验证。成功筛选出了具有高亲和活性的PCSK9单域抗体。
为此，在本发明的第一方面，本发明提出了一种能够特异性识别PCSK9的抗体或其抗原结合片段。根据本发明的实施例，所述抗体包括如SEQ ID NO:1、2和3或者与SEQ ID NO:1、2和3具有至少95％同一性的氨基酸序列所示的重链可变区CDR1、CDR2、CDR3序列。
GYTYSSNC(SEQ ID NO:1)。
IYIGGGST(SEQ ID NO:2)。
AVGCQGLVDFGY(SEQ ID NO:3)。
其中，重链可变区CDR1具有SEQ ID NO:1所示氨基酸序列或者与SEQ ID NO:1具有至少95％同一性的氨基酸序列；重链可变区CDR2具有SEQ ID NO:2所示氨基酸序列或者与SEQ ID NO:2具有至少95％同一性的氨基酸序列；重链可变区CDR3具有SEQ ID NO:3所示氨基酸序列或者与SEQ ID NO:3具有至少95％同一性的氨基酸序列。根据本发明实施例的抗体能够特异性结合PCSK9，抑制PCSK9的活性。
根据本发明的实施例，上述抗体还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述抗体含有重链框架区序列，所述重链框架区序列的至少一 部分来自于骆驼源抗体、鼠源抗体、人源抗体、灵长目源抗体或其突变体的至少之一。
根据本发明的实施例，所述重链框架区序列来自于骆驼源抗体。
根据本发明的实施例，所述重链框架区序列具有SEQ ID NO:4～15任一所示的氨基酸序列。
Figure PCTCN2020074624-appb-000001
根据本发明的实施例，所述重链框架区序列具有SEQ ID NO:8～15任一所示的氨基酸序列。发明人对SEQ ID NO:4～7所示氨基酸序列的重链框架区进行了某些氨基酸突变，获得的了SEQ ID NO:8～15所示氨基酸序列的重链框架区。发明人发现，SEQ ID NO:8～15所示氨基酸序列的重链框架区可以显著提高抗体的人源化程度，降低抗体免疫原性。
根据本发明的实施例，所述抗体具有如SEQ ID NO：16～25任一项所示氨基酸序列的重链可变区。
Figure PCTCN2020074624-appb-000002
Figure PCTCN2020074624-appb-000003
根据本发明的实施例，所述抗体具有如SEQ ID NO：17～25任一项所示氨基酸序列的重链可变区。发明人发现，具有17～25所示氨基酸序列的重链可变区的抗体的人源化更高，免疫原性更低。
根据本发明的实施例，所述抗体含有重链恒定区，所述重链恒定区的至少之一的至少一部分来自于鼠源抗体、人源抗体、灵长目源抗体或其突变体的至少之一。进而进一步提高了抗体的体内半衰期，提高了抗体的稳定性。
根据本发明的实施例，所述抗体的重链恒定区来自于人源IgG抗体或其突变体。
根据本发明的实施例，所述抗体的重链恒定区来自于人源IgG4。
根据本发明的实施例，所述抗体恒定区的全长序列如SEQ ID NO:26所示。
Figure PCTCN2020074624-appb-000004
Figure PCTCN2020074624-appb-000005
其中，PPCPSCP为铰链区序列，APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK为恒定CH2区，GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK为恒定CH3区。
根据本发明的实施例，所述抗体具有SEQ ID NO:27～36任一项所示氨基酸序列的重链。
Figure PCTCN2020074624-appb-000006
Figure PCTCN2020074624-appb-000007
Figure PCTCN2020074624-appb-000008
根据本发明实施例的上述具有SEQ ID NO:27～36任一项所示氨基酸序列重链的抗体，对PCSK9具有高亲和活性，且在体内的半衰期延长，稳定性更高。
根据本发明的实施例，所述抗体为小分子抗体。
根据本发明的实施例，所述小分子抗体包括单域抗体、Fab抗体、Fv抗体以及最小识别单位的至少之一。
根据本发明的实施例，所述抗体为单域抗体。根据本发明实施例的抗体作为单域抗体，对热和PH有较强的抵抗力，与抗原PCSK9的亲和力更高，还具有稳定性高、毒性低、可溶性强和易于在原核微生物中直接表达，经济性好等诸多优势。
在本发明的第二方面，本发明提出了一种核酸分子。根据本发明的实施例，所述核酸分子编码前面任一项所述的抗体或其抗原结合片段。
根据本发明的实施例，上述核酸分子还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述核酸分子为DNA。
根据本发明的实施例，所述核酸分子具有如SEQ ID NO:37～46任一项所示核苷酸序列。
Figure PCTCN2020074624-appb-000009
Figure PCTCN2020074624-appb-000010
Figure PCTCN2020074624-appb-000011
其中，SEQ ID NO:37所示核苷酸序列编码SEQ ID NO:16的重链可变区，SEQ ID NO:38所示核苷酸序列编码SEQ ID NO:17的重链可变区SEQ ID NO:39所示核苷酸序列编码SEQ ID NO:18的重链可变区，SEQ ID NO:40所示核苷酸序列编码SEQ ID NO:19的重链可变区， SEQ ID NO:,41所示核苷酸序列编码SEQ ID NO:20的重链可变区，SEQ ID NO:42所示核苷酸序列编码SEQ ID NO:21的重链可变区，SEQ ID NO:43所示核苷酸序列编码SEQ ID NO:22的重链可变区，SEQ ID NO:44所示核苷酸序列编码SEQ ID NO:23的重链可变区，SEQ ID NO:45所示核苷酸序列编码SEQ ID NO:24的重链可变区，SEQ ID NO:46所示核苷酸序列编码SEQ ID NO:25的重链可变区。
在本发明的第三方面，本发明提出了一种表达载体。根据本发明的实施例，所述表达载体携带前面所述的核酸分子。将根据本发明实施例的表达载体导入受体细胞后，在适于蛋白表达的条件下，表达前面所述的抗体，进而获得具有高PCSK9亲和活性的抗体。
根据本发明的实施例，上述表达载体还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述表达载体为原核表达载体。根据本发明实施例的抗体更容易在原核系统中进行表达，进而选择合适的原核表达载体，可进一步提高抗体在原核系统中的表达。
在本发明的第四方面，本发明提出了一种重组细胞。根据本发明的实施例，所述重组细胞携带前面所述的核酸分子，或者表达前面所述的抗体或其抗原结合片段。
根据本发明的实施例，上述重组细胞还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述重组细胞是通过将前面所述的表达载体引入至宿主细胞中而获得的。
根据本发明的实施例，通过电转导的方法将所述表达载体引入所述宿主细胞中。
根据本发明的实施例，所述重组细胞为原核细胞。
在本发明的第五方面，本发明提出了一种药物组合物。根据本发明的实施例，所述药物组合物含有前面所述的抗体，前面所述的核酸分子，前面所述的表达载体或前面所述的重组细胞。根据本发明实施例的药物组合物可特异性抑制PCSK9，用于有效治疗或预防PCSK9相关性疾病，副作用小、药效时间长。
在本发明的第六方面，本发明提出了前面所述的抗体、前面所述的核酸分子、前面所述的表达载体或前面所述的重组细胞、前面所述的药物组合物在制备药物中的用途，所述药物用于治疗或者预防PCSK9相关性疾病。所述药物用于治疗或预防PCSK9相关性疾病，具有副作用小、药效时间长的优势。
根据本发明的实施例，上述用途还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述药物用于治疗或预防高脂血症，高胆固醇血症，和/或动脉粥样硬化引起的病症，优选哺乳动物中的，优选人中的心血管疾病，中风或外周血管疾病。
在本发明的第七方面，本发明提出了一种检测PCSK9的试剂盒。根据本发明的实施例，所述试剂盒包括前面所述的抗体。根据本发明实施例的试剂盒可用于PCSK9的特异性检测
在本发明的第八方面，本发明提出了前面所述的抗体、核酸分子、表达载体或重组细胞在制备试剂盒中的用途，所述试剂盒用于检测PCSK9或者诊断PCSK9相关的疾病。
附图说明
图1是根据本发明实施例的Nest-PCR扩增骆驼重链抗体的VHH片段的凝胶电泳鉴定图；
图2是根据本发明实施例的PCR验证构建的噬菌体展示库中VHH片段的重组效率的凝胶电泳结果图；
图3是根据本发明实施例的淘洗PCSK9单域抗体单克隆ELISA筛选结果图；
图4是根据本发明实施例的淘洗PCSK9单域抗体单克隆PCR筛选结果图；
图5是根据本发明实施例的SDS-PAGE验证PCSK9单域抗体的表达和纯化的结果图；
图6是根据本发明实施例的ELISA验证纯化的PCSK9单域抗体的亲和力的结果图；
图7是根据本发明实施例的SPR检测VHH4亲和力的结果图；
图8是根据本发明实施例的SPR检测VHH-Z3亲和力的结果图；
图9是根据本发明实施例的SPR检测VHH-Z3-Fc亲和力的结果图；
图10是根据本发明实施例的PCSK9抗体的体外药效学验证结果图；
图11是根据本发明实施例的PCSK9抗体的体内药效学验证结果图。
具体实施方式
下面详细描述本发明的实施例，所述实施例的示例在附图中示出，其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
在对本发明描述的过程中，对于本文中有关的术语进行了解释和说明，这些解释和说明仅仅是为了方便对于方案的理解，并不能看做是对本发明保护方案的限制。
单域抗体
本文中，术语“单域抗体”是在骆驼血液中发现的天然缺失轻链的重链抗体，仅包括分子量较重的重链H链。其中，肽链的氨基端(N端)氨基酸序列变化很大，称为可变区(V区)，羧基端(C端)相对稳定，变化很小，称为恒定区(C区)。H链的V区称为VH。在可变区中某些区域氨基酸组成和排列顺序具有更高的变化程度，称为高变区(Hypervariable region，HVR)，高变区为抗原和抗体结合的位置，因此也称为决定簇互补区(complementarity-determining region，CDR)。重链可变区有三个CDR区。CDR1和CDR3比人稍长，CDR3在三级结构中向外凸出，因此，推测单域抗体比传统抗体有更高的抗原 结合的特异性和亲和力。
本发明利用CHO细胞表达的PCSK9抗原免疫骆驼，获得了高特异性的高亲和力的抗PCSK9的单域抗体(Nanobody,Nb)。利用该抗体能够与PCSK9抗原特异性结合，从而可以靶向性治疗或预防高血脂症和心脑血管疾病。
在一些实施方案中，本发明提供了一种能够特异性识别PCSK9的抗体或者抗原结合片段，所述抗体含有选自下列至少之一的CDR序列或与其具有至少95％同一性的氨基酸序列：重链可变区CDR序列：SEQ ID NO:1～3。在另一些实施方案中，本发明所提供的抗体或者抗原结合片段与上述重链相比，具有保守氨基酸取代。“抗原结合片段”是指保持特异性结合抗原(PCSK9)能力的抗体片段。“保守氨基酸取代”指的是氨基酸被另一氨基酸发生生物学上、化学上或者结构上相似的残基所取代。生物学上相似的指的是该取代不破坏PCSK9抗体或者与PCSK9抗原的生物学活性。结构上相似指的是氨基酸具有相似长度的侧链，如丙氨酸、甘氨酸或丝氨酸，或具有相似大小的侧链。化学相似性指的是氨基酸具有相同的荷电或者都是亲水或者疏水的。例如疏水残基异亮氨酸、缬氨酸、亮氨酸或者甲硫氨酸相互取代。或者用极性氨基酸例如用精氨酸取代赖氨酸、谷氨酸取代天冬氨酸、谷氨酰胺取代天冬酰胺，丝氨酸取代苏氨酸等等。
在一些实施方案中，本发明提供了一种抗体或抗原结合片段，所述抗体或抗原结合片段具有SEQ ID NO：16～25任一项所示氨基酸序列的重链可变区。发明人通过抗体序列比对数据库(NCBI、IMGT)可得到上述抗重链可变区序列的CDR区(如SEQ ID NO:1～3所示)。在另一些实施方案中，所述抗体或抗原结合片段的重链可变区序列与SEQ ID NO:16～25所示氨基酸序列相比，具有保守氨基酸取代。当然，这些保守氨基酸取代不会对抗体或者抗原结合片段的生物学功能带来改变。在一些具体方式中，这些保守氨基酸取代可以发生在重链可变区中除了CDR区之外的氨基酸上。
在一些优选方案中，本发明提供了一种抗PCSK9抗体，该抗体具有SEQ ID NO:27～36任一项所示氨基酸序列的重链。
在一些优选方案中，本发明提供了一种抗PCSK9单域抗体，该抗体具有SEQ ID NO:17～25任一项所示氨基酸序列的重链可变区或SEQ ID NO:28～36任一项所示氨基酸序列的重链。
核酸分子、表达载体、重组细胞
在制备或者获取这些抗体的过程中，可以利用表达这些抗体的核酸分子，与不同的载体连接，然后在不同细胞中表达，来获得相应抗体。
为此，本发明还提供了一种分离的核酸分子，所述核酸分子编码上述所述的抗体或抗原结合片段。
在一些实施方案中，所述分离核酸分子具有如SEQ ID NO:37～46任一项所示核苷酸序列。
在一些实施方案中，所述分离的核酸分子与上述SEQ ID NO:37～46所示的核苷酸序列至少具有90％以上的同源性，优选具有95％以上的同源性，更优选具有98％、99％以上的同源性。这些与SEQ ID NO:37～46所示核苷酸序列具有同源性的序列，能够表达与SEQ ID NO:16～25相似的氨基酸序列，从而能够与PCSK9抗原特异性结合，实现抗体的靶向性功能。
本发明还提供了一种表达载体，所述表达载体包含上述分离的核酸分子。在将上述分离的多核苷酸连接到载体上时，可以将多核苷酸与载体上的控制元件直接或者间接相连，只要这些控制元件能够控制多核苷酸的翻译和表达等即可。当然这些控制元件可以直接来自于载体本身，也可以是外源性的，即并非来自于载体本身。当然，多核苷酸与控制元件进行可操作地连接即可。本文中“可操作地连接”是指将外源基因连接到载体上，使得载体内的控制元件，例如转录控制序列和翻译控制序列等等，能够发挥其预期的调节外源基因的转录和翻译的功能。当然用来编码抗体重链的多核苷酸，可以分别独立的插入到不同的载体上，常见的是插入到同一载体上。常用的载体例如可以为质粒、噬菌体等等。例如Plasmid-X质粒。
本发明还提供了一种重组细胞，该重组细胞中包含有该表达载体。可以将表达载体导入到原核细胞中，构建获得重组细胞，然后利用这些重组细胞表达本发明提供的抗体或者抗原结合片段。通过该重组细胞进行培养，即可以获得相应抗体。
药物组合物、试剂盒及制药用途和在制备试剂盒中的用途。
本发明还提供了一种药物组合物，所述药物组合物包括上述所述的抗体或者抗原结合片段和药学可接受的载体。
本文提供的抗PCSK9抗体可以掺入适合受试者施用的药物组合物中。通常，这些药物组合物包括本文提供的抗PCSK9抗体以及药学上可接受的载体。“药学上可接受的载体”可以包括生理学上相容的任何和所有溶剂、分散介质、包衣、抗细菌剂和抗真菌剂、等渗剂和延迟吸收剂等等。具体实例可以是水、盐水、磷酸盐缓冲盐水、葡萄糖、甘油、乙醇等以及它们的组合物中的一种或多种。有许多情况下，药物组合物中包括等渗剂，例如糖类、多元醇(如甘露醇、山梨醇)或氯化钠等。当然药学上可接受的载体还可包括微量的辅助物质，例如润湿剂或乳化剂、防腐剂或缓冲剂，用来延长抗体的保存限期或效力。
例如，本发明的抗体可掺入适用于胃肠外施用(例如静脉内、皮下、腹膜内、肌肉内)的药物组合物中。这些药物组合物可以被制备成各种形式。例如液体、半固体和固体剂型等，包括但不限于液体溶液(例如，注射溶液和输注溶液)、分散剂或悬浮剂、片剂、丸剂、粉 末、脂质体和栓剂。典型的药物组合物为注射溶液或输注溶液形式。所述抗体可通过静脉输注或注射或肌肉内或皮下注射来施用。
当然，本文中的抗PCSK9抗体还可以根据需要被制成试剂盒或者其他诊断性试剂的一部分。根据本发明的实施例，本发明还提供了一种试剂盒，所述试剂盒包括上述PCSK9抗体。应用本发明提供的试剂盒，例如可以用于免疫印迹、免疫沉淀等涉及到利用PCSK9抗原和抗体特异性结合性能，来检测的试剂盒等。这些试剂盒可包含下列中的任意一种或多种：拮抗剂、抗PCSK9抗体或者药物参照材料；蛋白纯化柱；免疫球蛋白亲和纯化缓冲剂；细胞的测定稀释剂；说明书或者文献等。抗PCSK9抗体可被用于不同类型的诊断测试，例如可以在体外或者体内检测各种各样的疾病或者药物、毒素或者其他蛋白等的存在。例如可以通过对受试者的血清或者血液进行检测，用来测试相关疾病。这种相关疾病可包括PCSK9相关疾病，例如脂血症，高胆固醇血症，和/或动脉粥样硬化引起的病症，心血管疾病，中风或外周血管疾病等等。当然本文提供的抗体也可以用于上述疾病的放射免疫检测和放射免疫治疗等等。
在利用本发明所提供的抗PCSK9抗体治疗上述疾病时，可以将本发明提供的抗PCSK9抗体提供给受试者即可。为此，本发明提供了一种用于治疗上述疾病的方法，包括向有需要的受试者施用本发明所提供的的抗体或其抗原结合片段。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
本发明利用真核表达的PCSK9抗原免疫骆驼，通过流式分选亲和淋巴细胞，获得高质量的PCSK9免疫单域抗体抗体文库。将PCSK9抗原包被在酶标版上，利用噬菌体展示技术筛选PCSK9免疫单域抗体抗体文库，再将筛选出的单域抗体转化至大肠杆菌表达系统中进行大量表达，从而能够在比较短的时间里获得具有高亲和力的PCSK9的单克隆单域抗体株。
发明人根据噬菌体筛选的结果自主设计了VHH-Hu和VHH-Z1～8九株抗体。
实施例1 PCSK9单域抗体噬菌体展示文库构建
(1)PCSK9免疫骆驼
将1mg的PCSK9与等体积的弗氏佐剂混合至5mL，注射于骆驼颈部皮下3-5点，免疫前从骆驼的耳缘静脉采血。每月免疫一次，共免疫注射4次；每次免疫时，采取骆驼外周血10mL。采血时将骆驼头向一侧固定，对动物采血部位的皮肤先剃毛，75％酒精消毒，待 干燥后采血，用手指压迫颈静脉沟处，待血管怒张后，于采血部位消毒进针采血，采集血液10mL于15mL EDTA抗凝管中，立即连续、缓慢摇动，充分混合，置于冰上，运回实验室。
(2)血液淋巴细胞样品分离
对免疫前和每次免疫后采集的血液样本分离淋巴细胞，分离方法如下：
i.在15mL离心管中加入7mL淋巴细胞分离液Ficoll；
ii.在已加入抗凝剂(EDTA)的新鲜全血中加入等体积PBS(1×)或生理盐水，充分混匀；
iii.取加淋巴细胞分离液的15mL离心管，小心地缓慢转入另一已加入淋巴细胞分离液的15mL离心管中，并使上述混合液处于淋巴细胞分离液液面之上(即两种液体不要混合，保留清晰的界面)，3000g离心20min；
iv.用1mL移液枪将上清(血浆样品)小心转移到1.5mL细胞冻存管中，写上动物编号和血浆字样，放入带绳小布袋中，置液氮罐保存。
v.用1mL移液枪小心分离出白细胞层到一个15mL离心管中；加满PBS(1×)到15mL；用PBS(1×)清洗白细胞，离心(3000g离心20min)，小心倾倒掉上清，不要搅动管底的细胞团块，回收白细胞在剩余0.1-0.2mL PBS中。
vi.加5倍体积的RNA later，轻轻混溶细胞团块，分成2份到1.5mL细胞冻存管中，置液氮罐保存。
(3)总RNA提取，cDNA合成
取一份冻存的淋巴细胞，加入1mL Trizol，室温静置10min后，加入0.2mL氯仿，剧烈震荡，室温静置，待溶液分层(约10min)，12,000rpm离心后，收集上层水相，加入等体积的异丙醇，混匀，室温静置15min，待核酸沉淀，高速离心去上清，RNA沉淀加入1mL的75％乙醇(DEPC水配制)进行洗涤，高速离心去上清，控干水分后，RNA用无核酸酶的水溶解，分别取1μL用于浓度和纯度测定。
取1μg RNA，采用SuperScript TM III First-Strand Synthesis SuperMix(Invitrogen)试剂盒进行cDNA合成，逆转录引物用Oligo dT，合成cDNA在-20℃冻存；
(4)噬菌体展示文库构建
PCR扩增：
以上述合成的cDNA为模板，采用Nest-PCR扩增骆驼重链抗体的V区(VHH)，表1为Nest-PCR引物的名称及序列。
表1：骆驼VHH片段扩增使用的引物信息
Figure PCTCN2020074624-appb-000012
Figure PCTCN2020074624-appb-000013
PCR反应条件如下：
第一轮
cDNA            2μL
Mix             12.5μL
CALL001         0.5μL
CALL002/006/007 0.5μL
水  补足至25uL
反应条件：95℃,5min；94℃，1min；57℃,1min；72℃,1min per cycle；72℃,7min；扩增35循环。
第二轮
模板      40ng
Mix       25μL
VHH For(10uM)   1μL
VHH Back(10uM)  1μL
水  补足至50μl
反应条件：95℃，5min；94℃，45’；60℃，45’；72℃，45’每循环；72℃，7min；扩增25循环。
PCR反应结束后，用1.5％的琼脂糖凝胶电泳检测PCR产物，图1为nest-PCR的电泳结果，第一轮PCR的目的基因片段在700bp处，切胶回收目的条带，进行第二轮PCR，目的基因片段在500bp处，切胶回收目的条带，即VHH片段。
用NEB的限制性内切酶NotI和PstI分别对VHH片段和载体进行双酶切，反应体系如下：
载体酶切体系：
Figure PCTCN2020074624-appb-000014
Figure PCTCN2020074624-appb-000015
片段酶切体系：
Figure PCTCN2020074624-appb-000016
37℃，酶切过夜，琼脂糖凝胶电泳后，切胶回收；将载体和VHH片段的酶切产物混合，用NEB的连接酶在16℃连接过夜；
(5)噬菌体展示库的构建
连接产物经PCR Purification Kit(QIAGEN)纯化后，取1μL转化TG感受态细胞，37℃复苏2h，梯度稀释至101，102，103，分别取300μL涂布平板，37℃，过夜培养，计算克隆数，约105个克隆/平板。
采用上述相同的转化方法，大量转化，直到文库的克隆数达到107以上。将所有克隆用LB洗脱下，5,000g，离心5min，沉淀用2mL LB悬浮，加入等体积的30％甘油，-80冻存。
(6)文库多样性检测：
随机挑取(5)的克隆30个，作为模板，进行克隆PCR反应，用1.5％琼脂糖凝胶电泳检测PCR产物，如图2所示，构建的PCSK9单域抗体文库的重组率为100％。然后对其测序，分析PCSK9单域抗体抗体文库的多样性，测序结果显示，15个单克隆有13种氨基酸序列，表明构建的文库有较好的多样性。
(7)噬菌体扩增和拯救
采用辅助噬菌体对PCSK9单域抗体的噬菌体库进行扩增和拯救。将(5)保存的单克隆文库接入100mL培养基中培养至对数生长期，加入MOI为20的辅助噬菌体，室温，静置30min，低速离心后，沉淀用培养基悬起，接入300mL培养基中，培养过夜。次日，3,000离心30min，收集上清，加入PEG沉淀噬菌体，冰上静置30min，3,000离心30min，沉淀为PCSK9单域抗体噬菌体库，用PBS悬浮沉淀后，测定其滴度为2.9X 10 12 pfu/mL。
实施例2用噬菌体展示技术淘洗PCSK9单域抗体抗体
(1)亲和PCSK9单域抗体噬菌体库淘洗
取100ng PCSK9抗原包被ELISA板，4℃，过夜孵育。次日，加入拯救出的PCSK9单域抗体噬菌体，室温，孵育2h；PBST洗孔10次，加入100μL三乙胺，室温，孵育30min，收集的噬菌体即亲和淘洗获得的PCSK9单域抗体噬菌体库；取10μL感染TG细胞涂布平板，用于测定筛选后的克隆数测定，剩余筛选后的噬菌体的用于扩增。
(2)筛选后噬菌体的扩增和拯救
扩增和拯救方法同实施例1(7)，获得的PBS悬浮液即扩增的第一轮筛选后的噬菌体，置于4℃保存，并用于下一轮的筛选；按上述相同筛选步骤，逐次递减抗原量，筛选3-4轮。
(3)ELISA评价特异性抗体的富集程度
ELISA板包被100ng的PCSK9抗原，4℃，过夜；次日加2％的BSA室温封闭1h；实验组分别加入每轮淘洗后扩增的噬菌体，对照组加入等量野生型的噬菌体，室温，孵育2h；PBST洗10次，以去除没有结合的噬菌体；加入HRP标记的抗M13抗体，室温孵育1h；加入显色液，避光反应10-30min，测吸光值，吸光值随着淘洗次数逐渐上升，并在第三轮到第四轮淘洗时趋于稳定，表明特异性的抗体得到了富集。
(4)鉴定PCSK9特异性的单域抗体阳性克隆
ELISA板包被100ng的PCSK9抗原，4℃孵育过夜；取最后一轮筛选获得的噬菌体涂布的平板，随机挑取38个单克隆于1mL培养基中，37℃，培养至对数期，加入1mM IPTG诱导过夜；次日，离心收集菌沉，破碎后，5,000g离心15min，收集上清；同时取ELISA板，加2％的BSA室温封闭1h；实验组每孔加入单克隆破碎上清，对照组加入空白TG破碎上清，室温，孵育2h；PBST洗10次，加入鼠抗HA标签的抗体，室温1h；PBST洗3-5次，加入AP标记的抗鼠IgG抗体，室温1h；加入底物，反应10-20min，在酶标仪上读取吸光值；当吸光值与对照孔比值大于2.1(Base line)时，判定为阳性克隆；ELISA验证结果显示，获得30个阳性克隆(图3)；
(5)阳性克隆序列分析
提取(4)中获得的30个阳性克隆的DNA对插入片段进行PCR验证，如图4所示，经PCR验证为阳性的克隆进行测序分析。测序结果显示，获得两种核苷酸序列，对其氨基酸序列进行分析，其中一种序列具有典型的单域抗体的结构，即由框架区(FR1，FR2，FR3和FR4)和互补决定区(CDR1，CDR2和CDR3)构成。
这株单域抗体VHH4单克隆的氨基酸序列如下：
Figure PCTCN2020074624-appb-000017
框架区(FR1-FR4)和互补决定区(CDR1-CDR3)氨基酸序列：
FR1为QVQLQESGGGSVQAGGSLRLSCTVS(SEQ ID NO:4)，FR2为MGWFRQAPGKEHEGVAS(SEQ ID NO:5)，FR3为YYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYC(SEQ ID NO:6)，FR4为WDQGTQVTVSS(SEQ ID NO:7)，CDR1为GYTYSSNC(SEQ ID NO:1)，CDR 2的序列为IYIGGGST(SEQ ID NO:2)，CDR 3的序列为AVGCQGLVDFGY(SEQ ID NO:3)。
为提高单域抗体人源化程度，降低免疫原性，突变了VHH4四个框架区的某些氨基酸得到九个抗体序列VHH-Hu和VHHZ1～8。
PCSK9的抗体蛋白VHH-Hu，其氨基酸序列为：EVQLLESGGGLVQPGGSLRLSCAASGYTYSSNCMGWVRQAPGKGLEWVSSIYIGGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVGCQGLVDFGYWDQGTQVTVSS(SEQ ID NO:17)。其中，
框架区1的序列为EVQLLESGGGLVQPGGSLRLSCAAS(SEQ ID NO:8)，
框架区2的序列为MGWVRQAPGKGLEWVSS(SEQ ID NO:9)，
框架区3的序列为YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC(SEQ ID NO:10)，
框架区4的序列为WDQGTQVTVSS(SEQ ID NO:7)，
CDR1的序列为GYTYSSNC(SEQ ID NO:1)，
CDR2的序列为IYIGGGST(SEQ ID NO:2)，
CDR3的序列为AVGCQGLVDFGY(SEQ ID NO:3)。
PCSK9的抗体蛋白VHH-Z1，其氨基酸序列为：
EVQLLESGGGLVQPGGSLRLSCTVSGYTYSSNCMGWFRQAPGKEHEGVSSIYIGGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVGCQGLVDFGYWDQGTQVTVSS(SEQ ID NO:18)。其中，
框架区1的序列为EVQLLESGGGLVQPGGSLRLSCTVS(SEQ ID NO:11)，
框架区2的序列为MGWFRQAPGKEHEGVSS(SEQ ID NO:12)，
框架区3的序列为YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC(SEQ ID NO:10)，
框架区4的序列为WDQGTQVTVSS(SEQ ID NO:7)，
CDR1的序列为GYTYSSNC(SEQ ID NO:1)，
CDR2的序列为IYIGGGST(SEQ ID NO:2)，
CDR3的序列为AVGCQGLVDFGY(SEQ ID NO:3)。
PCSK9的抗体蛋白VHH-Z2，其氨基酸序列为：
EVQLLESGGGLVQPGGSLRLSCAASGYTYSSNCMGWFRQAPGKEHEGVSSIYIGGGSTYYADSVKGRFTISQDNSKNTLYLQMNSLRAEDTAVYYCAVGCQGLVDFGYWDQGTQVTVSS(SEQ ID NO:19)。其中，
框架区1的序列为EVQLLESGGGLVQPGGSLRLSCAAS(SEQ ID NO:8)，
框架区2的序列为MGWFRQAPGKEHEGVSS(SEQ ID NO:12)，
框架区3的序列为YYADSVKGRFTISQDNSKNTLYLQMNSLRAEDTAVYYC(SEQ ID NO:13)，
框架区4的序列为WDQGTQVTVSS(SEQ ID NO:7)，
CDR1的序列为GYTYSSNC(SEQ ID NO:1)，
CDR2的序列为IYIGGGST(SEQ ID NO:2)，
CDR3的序列为AVGCQGLVDFGY(SEQ ID NO:3)。。
PCSK9的抗体蛋白VHH-Z3，其氨基酸序列为：
EVQLLESGGGLVQPGGSLRLSCAASGYTYSSNCMGWFRQAPGKEHEGVASIYIGGGSTYYADSVKGRFTISQDNAKNTLYLQMNSLRAEDTAVYYCAVGCQGLVDFGYWDQGTQVTVSS(SEQ ID NO:20)。其中，
框架区1的序列为EVQLLESGGGLVQPGGSLRLSCAAS(SEQ ID NO:8)，
框架区2的序列为MGWFRQAPGKEHEGVAS(SEQ ID NO:5)，
框架区3的序列为YYADSVKGRFTISQDNAKNTLYLQMNSLRAEDTAVYYC(SEQ ID NO:14)，
框架区4的序列为WDQGTQVTVSS(SEQ ID NO:7)，
CDR1的序列为GYTYSSNC(SEQ ID NO:1)。
CDR2的序列为IYIGGGST(SEQ ID NO:2)。
CDR3的序列为AVGCQGLVDFGY(SEQ ID NO:3)。
PCSK9的抗体蛋白VHH-Z4，其氨基酸序列为：
EVQLLESGGGLVQPGGSLRLSCAASGYTYSSNCMGWFRQAPGKEHEGVSSIYIGGGSTYYADSVKGRFTISQDNAKNTLYLQMNSLRAEDTAVYYCAVGCQGLVDFGYWDQGTQVTVSS(SEQ ID NO:21)。其中，
框架区1的序列为EVQLLESGGGLVQPGGSLRLSCAAS(SEQ ID NO:8)，
框架区2的序列为MGWFRQAPGKEHEGVSS(SEQ ID NO:12)，
框架区3的序列为YYADSVKGRFTISQDNAKNTLYLQMNSLRAEDTAVYYC(seq id no:14)，
框架区4的序列为WDQGTQVTVSS(SEQ ID NO:7)，
CDR1的序列为GYTYSSNC(SEQ ID NO:1)，
CDR2的序列为IYIGGGST(SEQ ID NO:2)，
CDR3的序列为AVGCQGLVDFGY(SEQ ID NO:3)。
PCSK9的抗体蛋白VHH-Z5，其氨基酸序列为：
EVQLLESGGGLVQPGGSLRLSCAASGYTYSSNCMGWFRQAPGKEHEGVSSIYIGGGSTYYADSVKGRFTISQDNSKNTVYLQMNSLRAEDTAVYYCAVGCQGLVDFGYWDQGTQVTVSS(SEQ ID NO:22)。其中，
框架区1的序列为EVQLLESGGGLVQPGGSLRLSCAAS(SEQ ID NO:8)，
框架区2的序列为MGWFRQAPGKEHEGVSS(SEQ ID NO:12)，
框架区3的序列为YYADSVKGRFTISQDNSKNTVYLQMNSLRAEDTAVYYC(SEQ ID NO:15)，
框架区4的序列为WDQGTQVTVSS(SEQ ID NO:7)，
CDR1的序列为GYTYSSNC(SEQ ID NO:1)，
CDR2的序列为IYIGGGST(SEQ ID NO:2)，
CDR3的序列为AVGCQGLVDFGY(SEQ ID NO:3)。
PCSK9的抗体蛋白VHH-Z6，其氨基酸序列为：
EVQLLESGGGLVQPGGSLRLSCTVSGYTYSSNCMGWFRQAPGKEHEGVSSIYIGGGSTYYADSVKGRFTISQDNSKNTLYLQMNSLRAEDTAVYYCAVGCQGLVDFGYWDQGTQVTVSS(SEQ ID NO:23)。其中，
框架区1的序列为EVQLLESGGGLVQPGGSLRLSCTVS(SEQ ID NO:11)，
框架区2的序列为MGWFRQAPGKEHEGVSS(SEQ ID NO:12)，
框架区3的序列为YYADSVKGRFTISQDNSKNTLYLQMNSLRAEDTAVYYC(SEQ ID NO:13)，
框架区4的序列为WDQGTQVTVSS(SEQ ID NO:7)，
CDR1的序列为GYTYSSNC(SEQ ID NO:1)，
CDR2的序列为IYIGGGST(SEQ ID NO:2)，
CDR3的序列为AVGCQGLVDFGY(SEQ ID NO:3)。
PCSK9的抗体蛋白VHH-Z7，其氨基酸序列为：
EVQLLESGGGLVQPGGSLRLSCTVSGYTYSSNCMGWFRQAPGKEHEGVSSIYIGGGSTYYADSVKGRFTISQDNAKNTLYLQMNSLRAEDTAVYYCAVGCQGLVDFGYWDQGTQVTVSS(SEQ ID NO:24)。其中，
框架区1的序列为EVQLLESGGGLVQPGGSLRLSCTVS(SEQ ID NO:11)，
框架区2的序列为MGWFRQAPGKEHEGVSS(SEQ ID NO:12)，
框架区3的序列为YYADSVKGRFTISQDNAKNTLYLQMNSLRAEDTAVYYC(SEQ ID NO:14)，
框架区4的序列为WDQGTQVTVSS(SEQ ID NO:7)，
CDR1的序列为GYTYSSNC(SEQ ID NO:1)，
CDR2的序列为IYIGGGST(SEQ ID NO:2)，
CDR3的序列为AVGCQGLVDFGY(SEQ ID NO:3)。
PCSK9的抗体蛋白VHH-Z8，其氨基酸序列为：
EVQLLESGGGLVQPGGSLRLSCAASGYTYSSNCMGWFRQAPGKEHEGVSSIYIGGGSTYYADSVKGRFTISQDNAKNTLYLQMNSLRAEDTAVYYCAVGCQGLVDFGYWDQGTQVTVSS(SEQ ID NO:25)。其中，
框架区1的序列为EVQLLESGGGLVQPGGSLRLSCAAS(SEQ ID NO:8)，
框架区2的序列为MGWFRQAPGKEHEGVSS(SEQ ID NO:12)，
框架区3的序列为YYADSVKGRFTISQDNAKNTLYLQMNSLRAEDTAVYYC(SEQ ID NO:14)，
框架区4的序列为WDQGTQVTVSS(SEQ ID NO:7)，
CDR1的序列为GYTYSSNC(SEQ ID NO:1)，
CDR2的序列为IYIGGGST(SEQ ID NO:2)，
CDR3的序列为AVGCQGLVDFGY(SEQ ID NO:3)。
提高抗体的体内半衰期，融合了人IgG4 Fc区域，得到了VHH-Hu-Fc和VHHZ1～8-Fc九个Fc融合抗体，序列如下所示。
Figure PCTCN2020074624-appb-000018
Figure PCTCN2020074624-appb-000019
其中：
铰链区Hinge region的序列为:PPCPSCP
恒定区CH2区的序列为：
Figure PCTCN2020074624-appb-000020
恒定区CH3区的序列为：
Figure PCTCN2020074624-appb-000021
实施例3 PCSK9单域抗体的诱导表达和纯化
(1)PCSK9单域抗体表达菌构建
首先将PCSK9单域抗体单克隆转接培养基，37℃，过夜培养；次日，用E.Z.N.A.
Figure PCTCN2020074624-appb-000022
Plasmid Mini Kit I(OMEGA)提取质粒，琼脂糖凝胶电泳并测定浓度后，将含有PCSK9单域抗体序列的质粒转化入表达菌HB2151中，涂布平板，37℃，培养过夜。
(2)PCSK9单域抗体的诱导表达
次日，从平板挑取5个克隆进行PCR验证质粒是否转入表达菌株；挑取阳性克隆37℃培养至OD600为0.6-0.8，加入IPTG进行诱导表达。离心菌液，收集菌体沉淀，用裂解缓冲液重悬沉淀，超声破碎菌体，离心收集破碎后的菌体上清。
(3)PCSK9单域抗体的纯化
通过Ni柱亲和纯化获得PCSK9单域抗体。Ni柱先用超纯水清洗，然后用裂解液清洗；将上述PCSK9单域抗体表达菌的破碎上清以1mL/min的流速加入Ni柱；用5倍柱体积的亲和A液(20mM咪唑)洗去杂蛋白，再用等体积的亲和B液(250mM咪唑)洗脱目的蛋白，并收集洗脱液；最后15％的SDS-PAGE监测PCSK9单域抗体的表达和纯化情况(图5)。
(4)VHH-Fc表达载体构建
通过PCR扩增目的基因或外包合成基因序列；将目的基因插入对应表达载体pCDNA3.4；测序得到含有正确序列的质粒；质粒放大生产传递下游表达
(5)哺乳细胞表达
HEK293细胞-用293无血清CD培养基(北京义翘，货号SMM 293-TI)传代培养HEK293细胞,将要表达的质粒VHH4-Fc和VHH-Z3-Fc与转染试剂TF2(北京义翘，货号STF02)混合后加入细胞中，转染后第1、3、5天添加293无血清加料液(北京义翘，货号M293-SUPI-100)。摇瓶培养条件：5％CO 2，温度37℃，摇床转速175rpm。
(6)纯化
培养料液6000rpm离心20min后，用滤器过滤，取上清。
a、层析柱预处理：ProteinA亲和层析柱用超纯水冲洗，然后用平衡缓冲液平衡；
b、上样与平衡：料液上样ProteinA亲和层析柱，上样完毕后用平衡缓冲液淋洗至基线平稳；
c、洗脱：用Elution3.0(0.1M的柠檬酸)洗脱，收集洗脱峰,用2M Tris中和；
d、ProteinA亲和层析柱再生；
e、纯化样品用脱盐至适当的缓冲液中；
f、纯化样品无菌过滤后留样送检QC。
实施例4 PCSK9抗体的亲和力测定
(1)ELISA法分析PCSK9单域抗体的亲和力
实验组以100ng的PCSK9蛋白，对照组用未诱导的质粒表达的蛋白和BSA包被酶标板，4℃孵育过夜；次日加2％的BSA室温封闭1h；取纯化的PCSK9抗体分别加入对照组和实验组，空白组加入PBS，室温，孵育2h；PBST洗10次，加入二抗，室温1h；加入底物，反应10-20min，在酶标仪上读取吸光值。ELISA检测结果(图6)显示，PCSK9单域抗体对PCSK9具有较好的亲和力，结合活性远远高于对照组。
(2)SPR分析PCSK9单域抗体的结合常数
活化芯片后，加入500nM的PCSK9抗原进行反应；加入150μL的1M乙醇胺盐酸洗掉残留的活性羧基基团；将PCSK9抗体进行梯度稀释后加入360μL，速率为25μL/min，结合120s，解离400s；获得数据后，进行结果处理，结果如下图7、8、9显示，表示抗体的结合位点被抗原占领时的信号强度，KD(M)为抗原抗体相互作用的解离常数，表明PCSK9各个抗体与PCSK9抗原的相互作用良好，具有继续开发的价值。
实施例5 PCSK9抗体在癌细胞模型上的体外药效验证实验
将培养好的Huh7和HepG2细胞在96孔板(美国康宁公司)里以5×10 5个细胞/孔的细胞密度(用新鲜的含10％胎牛血清的DMEM重悬细胞)铺板。37度培养18-20h后，细胞用D-PBS(美国Gibco品牌)清洗后转至无血清培养系统。实验总共设计五组：第一组为空白对照(不加hPCSK9和抗体)。第二组为阴性对照(只添加hPCSK9，浓度为0.08μM。随后将预热37℃的hPCSK9(浓度为0.08μM)蛋白和其他两组抗体(VHH-Z3和VHHZ3-Fc，浓度分别设置为0.75μM，0.375μM，和1.5μM)加入到上述细胞当中。37℃培养1h后加入10μL的LDL-BIODIPY(美国英潍捷基)到细胞中(浓度为6μg/mL)和细胞进一步37℃培养3h。用D-PBS(美国Gibco品牌)三遍清洗后用荧光酶标仪M1000 PRO(Tecan， 瑞士)检测相对荧光强度。荧光强度可以反映低密度脂蛋白LDL的代谢水平。结果如图10所示。在两组肝癌细胞模型中，加VHH4及VHH-Z3和VHHZ3-Fc抗体均可阻断PCSK9与LDLR的结合，从而促进细胞表面的LDLR吸收LDLC。
实施例6 PCSK9抗体在转基因大鼠上的体内药效学验证实验
动物药效学实验分成七个组，每组六只大鼠，其中有五个组为hPCSK9转基因鼠(Tg+鼠)组和两个正常对照大鼠组。hPCSK9转基因大鼠是通过在正常大鼠的受精卵基因组随机插入hPCSK9基因来实现的。成功转基因的大鼠通过PCR验证hPCSK9基因的正确插入。这些Tg+大鼠经过8周的高脂饮食诱导产生高血脂症状。转基因大鼠五个组中，前四组继续高脂饮食，且分别在尾静脉注射PBS、市面在售的瑞百安单抗(Evolocumab)、VHH-Z3和VHH-Z3-Fc(注射剂量为20mg/Kg)。它们依次被命名为转基因高脂空白组、转基因高脂阳性组、转基因高脂VHH-Z3组和转基因高脂VHH-Z3-Fc组，剩余一组转基因大鼠以正常饮食喂养。同时设置两个正常大鼠的对照组。第一组是非转基因正常饮食组，第二组为非转基因高脂饮食喂养，注射了市售瑞百安单抗，命名为非转基因高脂阳性组。老鼠分别在0到7天实施注射，在19天进行测试。血液样本收集和总胆固醇(CHOL)和肝脏低密度脂蛋白水平都通过自动生化分析仪进行测定(Bs-600，迈瑞品牌，中国)，结果如图11所示。与对照组相比，高血脂症hPCSK9大鼠给予阳性药物后可明显降低CHOL及LDL-C的水平并维持至实验终点。供试药物VHH-Z3-Fc对hPCSK9高血脂症模型大鼠的CHOL及LDL-C水平同样具有明显的降低作用，并且在给药后第2天至13天间，其降脂稍好于于阳性药物组。供试药物VHH-Z3在整个给药期间对hPCSK9高血脂症模型大鼠的CHOL及LDL-C水平没有明显的影响
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (27)

  1. 一种能够特异性识别PCSK9的抗体或其抗原结合片段，其特征在于，所述抗体包括如SEQ ID NO:1、2和3或者与SEQ ID NO:1、2和3具有至少95％同一性的氨基酸序列所示的重链可变区CDR1、CDR2、CDR3序列。
  2. 根据权利要求1所述的抗体或其抗原结合片段，其特征在于，所述抗体含有重链框架区序列，所述重链框架区序列的至少一部分来自于骆驼源抗体、鼠源抗体、人源抗体、灵长目源抗体或其突变体的至少之一。
  3. 根据权利要求2所述的抗体或其抗原结合片段，其特征在于，所述重链框架区序列来自于骆驼源抗体。
  4. 根据权利要求2所述的抗体或其抗原结合片段，其特征在于，所述重链框架区序列具有SEQ ID NO:4～15任一所示的氨基酸序列；
    优选地，所述重链框架区序列具有SEQ ID NO:8～15任一所示的氨基酸序列；
  5. 根据权利要求1所述的抗体或其抗原结合片段，其特征在于，所述抗体具有如SEQ ID NO：16～25任一项所示氨基酸序列的重链可变区；
    优选地，所述抗体具有如SEQ ID NO：17～25任一项所示氨基酸序列的重链可变区。
  6. 根据权利要求1所述的抗体或其抗原结合片段，其特征在于，所述抗体含有重链恒定区，所述重链恒定区的至少一部分来自于鼠源抗体、人源抗体、灵长目源抗体或其突变体的至少之一。
  7. 根据权利要求1所述的抗体或其抗原结合片段，其特征在于，所述抗体的重链恒定区来自于人源IgG抗体或其突变体。
  8. 根据权利要求1所述的抗体或其抗原结合片段，其特征在于，所述抗体的重链恒定区来自于人源IgG4。
  9. 根据权利要求8所述的抗体或其抗原结合片段，其特征在于，所述抗体恒定区的全长序列如SEQ ID NO:26所示。
  10. 根据权利要求9所述的抗体或其抗原结合片段，其特征在于，所述抗体具有SEQ ID NO:27～36任一项所示氨基酸序列的重链。
  11. 根据权利要求1所述的抗体或其抗原结合片段，其特征在于，所述抗体为小分子抗体。
  12. 根据权利要求11所述的抗体或其抗原结合片段，其特征在于，所述小分子抗体包括单域抗体、Fab抗体、Fv抗体以及最小识别单位的至少之一。
  13. 根据权利要求1所述的抗体或其抗原结合片段，其特征在于，所述抗体为单域抗 体。
  14. 一种核酸分子，其特征在于，所述核酸分子编码权利要求1～13任一项所述的抗体或其抗原结合片段。
  15. 根据权利要求14所述的核酸分子，其特征在于，所述核酸分子为DNA。
  16. 根据权利要求15所述的核酸分子，其特征在于，所述核酸分子具有如SEQ ID NO:23～31任一项所示核苷酸序列。
  17. 一种表达载体，其特征在于，携带权利要求14～16任一项所述的核酸分子。
  18. 根据权利要求17所述的表达载体，其特征在于，所述表达载体为原核表达载体。
  19. 一种重组细胞，其特征在于，所述重组细胞携带权利要求14～16任一项所述的核酸分子，或者表达权利要求1～13任一项所述的抗体或其抗原结合片段。
  20. 根据权利要求19所述的重组细胞，其特征在于，所述重组细胞是通过将权利要求17或18所述的表达载体引入至宿主细胞中而获得的。
  21. 根据权利要求20所述的重组细胞，其特征在于，通过电转导的方法将所述表达载体引入所述宿主细胞中。
  22. 根据权利要求21所述的重组细胞，其特征在于，所述重组细胞为原核细胞。
  23. 一种药物组合物，其特征在于，含有权利要求1～13任一项所述的抗体，权利要求14～16任一项所述的核酸分子，权利要求17～18任一项所述的表达载体或权利要求19～22任一项所述的重组细胞。
  24. 权利要求1～13任一项所述的抗体、权利要求14～16任一项所述的核酸分子、权利要求17～18任一项所述的表达载体或权利要求19～22任一项所述的重组细胞、权利要求23所述的药物组合物在制备药物中的用途，所述药物用于治疗或者预防PCSK9相关性疾病。
  25. 根据权利要24所述的用途，其特征在于，所述药物用于治疗或预防高脂血症，高胆固醇血症，和/或动脉粥样硬化引起的病症，优选哺乳动物中的，优选人中的心血管疾病，中风或外周血管疾病。
  26. 一种检测PCSK9的试剂盒，其特征在于，包括权利要求利要求1～13任一项所述的抗体。
  27. 权利要求1～13任一项所述的抗体、权利要求14～16任一项所述的核酸分子、权利要求17～18任一项所述的表达载体或权利要求19～22任一项所述的重组细胞在制备试剂盒中的用途，所述试剂盒用于检测PCSK9或者诊断PCSK9相关的疾病。
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