CN106084058A - Anti-human PCSK9 monoclonal antibody - Google Patents

Anti-human PCSK9 monoclonal antibody Download PDF

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CN106084058A
CN106084058A CN201610540921.6A CN201610540921A CN106084058A CN 106084058 A CN106084058 A CN 106084058A CN 201610540921 A CN201610540921 A CN 201610540921A CN 106084058 A CN106084058 A CN 106084058A
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antibody
pcsk9
antigen
ldl
binding portion
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CN106084058B (en
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刘方杰
耿树生
李锋
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Beijing Mabworks Biotech Co Ltd
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Beijing Mabworks Biotech Co Ltd
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    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • G—PHYSICS
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    • G01—MEASURING; TESTING
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
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    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/567—Framework region [FR]
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96433—Serine endopeptidases (3.4.21)

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Abstract

The invention belongs to antibody art, be specifically related to anti-human PCSK9 monoclonal antibody, and described antibody is for the purposes prepared the lipoprotein levels reduced in blood, prevent or treat the medicine of cardiovascular disease or imbalance, thrombosis or imbalance.The anti-human PCSK9 monoclonal antibody affinity of the present invention is higher, has a good application prospect.

Description

Anti-human PCSK9 monoclonal antibody
Technical field
The invention belongs to antibody art, be specifically related to anti-human PCSK9 monoclonal antibody, and described antibody is used for preparing fall Lipoprotein levels in low blood, prevent or treat the medicine of cardiovascular disease or imbalance, thrombosis or imbalance Purposes.
Background technology
Proprotein convertase subtilisin/kexin9 (Proprotein Convertase Subtilisin/ Kexin type 9, PCSK9) it is a unique proprotein convertases belonging to serine protease K subclass.This front albumen converts Enzyme (PCSK9) gene mapping is in human chromosomal 1p32.3, and total length is 29kb, is made up of 12 exons, and coding region is about 2kb, coding comprises the protein of 692 amino acid residues.PCSK9 mainly by signal peptide, predomain, catalyst structure domain, and Carboxy-terminal domains forms.PCSK9 precursor protein synthesizes a kind of solubility proenzyme, i.e. PCSK9 proenzyme (apo-in endoplasmic reticulum PCSK9,74KD), endoplasmic reticulum or Golgi body occur at apo-PCSK9 151-152 residue autocatalysis division release Propetide (14KD) forms maturation protein enzyme (60KD) and is secreted in blood.PCSK9 mainly expresses in liver, small intestinal, but only Can be secreted in blood at the PCSK9 of liver expression.
Serum low-density LP level is to weigh the leading indicator of blood lipid level, and it is atherosclerotic that LDL raises Cardiopathic Major Risk Factors, can induce and promote atherosclerotic development.Numerous researchs show that PCSK9 can be with The low-density lipoprotein cholesterol receptor (low-density lipoprotein receptor, LDL-R) of cell surface combines And its internalization is guided to lysosomal degradation, suppress it to be recycled to surface of hepatocytes, thus weaken in liver metabolism blood plasma low The ability of density lipoprotein-cholesterol (LDL-c).Epidemiological study finds, PCSK9 gene different parts base mutation can be led Causing two kinds of distinct biological effects, one is the sudden change of gain-of-function type, and another kind is Loss-of-function sudden change.Function obtains Obtain type sudden change and can strengthen the ability of degraded hepatocyte LDLR, reduce so that the LDL-c in blood removes, cause hypercholesterolemia The generation of mass formed by blood stasis.Loss-of-function sudden change can destroy the normal function of PCSK9, causes surface of hepatocytes LDLR to increase, so that Obtain LDL-c in blood and remove increase.Research shows, this low-level blood caused because of PCSK9 gene function defect LDL-c can significantly reduce the sickness rate of atherosclerosis and coronary heart disease.Genetics research is further discovered that at PCSK9 egg In the white crowd lacked completely, their internal low-density lipoprotein white level is only 1/10th of general population, and this feelings Their health is not had any impact by condition.This explanation should not exist any by the function of Drug inhibition PCSK9 Side effect.Therefore, reduce the purpose of LDL-c by suppressing PCSK9, become the focus of recent researches.
In the research and development of numerous PCSK9 inhibitor, anti-PCSK9 monoclonal antibody gets most of the attention.The large-scale world of current many families Drugmaker all at active development for the monoclonal antibody medicine of huPCSK9, wherein Evolocumab and Sai Nuofei of Amgen/regeneration The Alirocumab of unit has completed III clinical trial phase.III phase clinical data shows, with standard treatment is compared, and both antibody All can reduce the LDL-c of 61% more, within 1 year, reduce by 50% cardiovascular event, long-term efficacy is had an optimistic view of.
Human antibody is the Main way of therapeutic antibodies development, and the full people source that appears as of phage antibody library technique resists The preparation of body provides good technology platform.Phage antibody library technique uses PCR method amplification in vitro people antibody VH and VL Portion gene, then by random for VH and VL recombinant clone to Vector for Phage Display.It is presented on the antibody energy of phage surface Enough interact with immobilised targeting antigen in vitro, remove non-specific binding antibody, then eluting by cyclic washing And collect the phage being combined with antigen, this phage ehec infection again, make specific phage be enriched with.? Worked by DNA sequencing afterwards, it is thus achieved that the antibody sequence specific binding with targeting antigen.Phage antibody library technique simulates body Interior immune selection and the process of affinity matured antibody, it is not necessary to through hybridoma technology, even without through exempting from Epidemic disease process can obtain specific and high-affinity various Antibody molecule fragments, substantially reduces the research and development time of antibody.
This area still lacks the anti-human PCSK9 human antibody with more high-affinity.
Summary of the invention
The present invention first filters out the monoclonal antibody of anti-huPCSK9 from naive phage antibody library rapidly, then should With the method for the complete synthesis phage antibody library building low capacity rite-directed mutagenesis, the weight of antagonist on the basis of the antibody filtered out Chain variable region CDR1,2,3 region mutations are built storehouse and are carried out the external affinity maturation of antibody, it is thus achieved that the antibody of high-affinity, thus Complete the present invention.
A first aspect of the present invention relates to anti-PCSK9 antibody or its antigen-binding portion thereof, and it includes selected from such as next group CDR region:
CDR1, CDR2, CDR3 district, variable region of heavy chain comprises the sequence as shown in SEQ ID NO:1,2,3 respectively, and light chain can Become CDR1, CDR2, CDR3 district of district and comprise the sequence as shown in SEQ ID NO:4-6 respectively；
8D8F VH CDR1:SGFAFGGYAMN (SEQ ID NO:1)
8D8F VH CDR2:TISGSGGSTN (SEQ ID NO:2)
8D8F VH CDR3:AKDSNWGNFDL (SEQ ID NO:3)
8D8F VL CDR1:KSSESVMYRRNARNFLG (SEQ ID NO:4)
8D8F VL CDR2:WASTRESGVPDR (SEQ ID NO:5)
8D8F VL CDR3:QQYYTHPYT (SEQ ID NO:6)
In one embodiment of the invention, described anti-PCSK9 antibody or its antigen-binding portion thereof, wherein:
The variable region of heavy chain of described anti-PCSK9 antibody comprises the CDR1-that aminoacid sequence is SEQ ID NO:1-3 CDR3；
And/or
The variable region of light chain of described anti-PCSK9 antibody comprises the CDR1-that aminoacid sequence is SEQ ID NO:4-6 CDR3。
In one embodiment of the invention, the variable region of heavy chain FR1 of described antibody or its antigen-binding portion thereof, FR2, FR3, FR4 district comprises the sequence as shown in SEQ ID NO:7-10 respectively, or comprises the homogeneity with above-mentioned sequence and be more than 70%, it is greater than the sequence of 75%, 80%, 85%, 90%, 95%, 99%.
VH FR1:EVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO:7)
VH FR2:WVRQAPGKGLDWVS (SEQ ID NO:8)
VH FR3:
YADSVKGRFIISRDSSKHTLYLQMNSLRAEDTAVYYC(SEQ ID NO:9)
VH FR4:WGRGTLVTVSS (SEQ ID NO:10)
In one embodiment of the invention, the variable region of light chain FR1 of described antibody or its antigen-binding portion thereof, FR2, FR3, FR4 district comprises the sequence as shown in SEQ ID NO:11-14 respectively, or comprises the homogeneity with above-mentioned sequence and be more than 70%, it is greater than the sequence of 75%, 80%, 85%, 90%, 95%, 99%.
VL FR1:DIVMTQSPDSLAVSLGERATINC (SEQ ID NO:11)
VL FR2:WYQQKPGQPPNLLIY (SEQ ID NO:12)
VL FR3:FSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO:13)
VL FR4:FGQGTKLEIK (SEQ ID NO:14)
In one embodiment of the invention, the sequence of the variable region of heavy chain of described antibody or its antigen-binding portion thereof Row are as shown in SEQ ID NO:15.Underscore part is CDR sequence.
EVQLVESGGGLVQPGGSLRLSCAASGFAFGGYAMNWVRQAPGKGLDWVSTISGSGGSTNYADSVKGRFIISRDSSKH TLYLQMNSLRAEDTAVYYCAKDSNWGNFDLWGRGTLVTVSS(SEQ ID NO:15)
In one embodiment of the invention, the sequence of the variable region of light chain of described antibody or its antigen-binding portion thereof Row are as shown in SEQ ID NO:16.Underscore part is CDR sequence.
DIVMTQSPDSLAVSLGERATINCKSSESVMYRRNARNFLGWYQQKPGQPPNLLIYWASTRESGVPDRFSGSGSGTDF TLTISSLQAEDVAVYYCQQYYTHPYTFGQGTKLEIK(SEQ ID NO:16)
In one embodiment of the invention, the CH of described antibody or its antigen-binding portion thereof derives from People.
In one embodiment of the invention, the CH of described antibody or its antigen-binding portion thereof is selected from source IgG, IgM, IgE, IgD and IgA in people.
In one embodiment of the invention, the constant region of light chain of described antibody or its antigen-binding portion thereof is selected from source IgG1, IgG2, IgG3 and IgG4 in people.
In one embodiment of the invention, the CH of described antibody or its antigen-binding portion thereof is next Coming from the IgG1 of people, the aminoacid sequence of described IgG1 CH is such as shown in SEQ ID NO:19.
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPG(SEQ ID NO:19)
The nucleotide sequence of coding IgG1 CH
In one embodiment of the invention, the constant region of light chain of described antibody or its antigen-binding portion thereof derives from People.
In one embodiment of the invention, the constant region of light chain of described antibody or its antigen-binding portion thereof is for deriving from κ or the λ type of people.
In one embodiment of the invention, the constant region of light chain of described antibody or its antigen-binding portion thereof is next Coming from the κ type of people, the aminoacid sequence of described κ type constant region of light chain is such as shown in SEQ ID NO:21.
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:21)
The nucleotide sequence of coding κ type constant region of light chain:
In one embodiment of the invention, described antibody be whole antibody, bi-specific antibody, scFv, Fab, Fab', F(ab')2Or Fv.
In one embodiment of the invention, described antibody is human antibody.
In one embodiment of the invention, described PCSK9 is people PCSK9.
In one embodiment of the invention, described anti-PCSK9 antibody or its antigen-binding portion thereof, wherein, described Anti-PCSK9 antibody with less than about 10-5M, the most about 10-6M、10-7M、10-8M、10-9M or 10-10The K of M or lessD In conjunction with PCSK9 albumen.
The invention still further relates to nucleic acid molecules, its contain the coding anti-PCSK9 antibody of any one of first aspect present invention or its Antigen-binding portion thereof or the sequence of its fragment；Preferably, described nucleic acid molecules has the nucleotides sequence shown in SEQ ID NO:17 Nucleotide sequence shown in row and/or SEQ ID NO:18.
In one embodiment of the invention, described nucleic acid molecules contains the anti-of coding any one of first aspect present invention CDR region (such as CDR1, CDR2, CDR3) sequence of the variable region of heavy chain of PCSK9 antibody or its antigen-binding portion thereof.
In one embodiment of the invention, described nucleic acid molecules contains the anti-of coding any one of first aspect present invention CDR region (such as CDR1, CDR2, CDR3) sequence of the variable region of light chain of PCSK9 antibody or its antigen-binding portion thereof.
In one embodiment of the invention, described nucleic acid molecules contains the anti-of coding any one of first aspect present invention The sequence of the variable region of heavy chain of PCSK9 antibody.In specific embodiments of the present invention, described nucleic acid molecules contains such as SEQ ID Sequence shown in NO:17.
In one embodiment of the invention, described nucleic acid molecules contains the anti-of coding any one of first aspect present invention The sequence of the variable region of light chain of PCSK9 antibody.In specific embodiments of the present invention, described nucleic acid molecules contains such as SEQ ID Sequence shown in NO:18.
The invention still further relates to carrier, it contains the nucleic acid molecules of any one of the present invention.
The invention still further relates to host cell, its nucleic acid molecules containing any one of the present invention or carrier.
Another aspect of the invention relates to a kind of conjugate, and it includes antibody or its Fab and coupling portion Point, wherein, described antibody is the anti-PCSK9 antibody described in any one of the present invention or its antigen-binding portion thereof, described coupling moiety For detectable labelling；Preferably, described coupling moiety be radiosiotope, fluorescent material, luminescent substance, coloring matter or Enzyme.
Another aspect of the invention relates to a kind of test kit, and it includes the anti-PCSK9 antibody according to any one of the present invention Or its antigen-binding portion thereof, or include the conjugate of the present invention；
Preferably, described test kit also includes second antibody, anti-PCSK9 antibody described in its specific recognition or its antigen knot Close part；Optionally, described second antibody also includes detectable labelling, such as radiosiotope, fluorescent material, shiner Matter, coloring matter or enzyme.
Another aspect of the invention relate to the anti-PCSK9 antibody described in any one of the present invention or its antigen-binding portion thereof or The conjugate of present invention purposes in preparing test kit, described test kit for detect PCSK9 existence in the sample or its Level.
The invention still further relates to compositions (such as pharmaceutical composition), it contains the anti-of any one of first aspect present invention PCSK9 antibody or its antigen-binding portion thereof, the nucleic acid molecules of any one of the present invention, carrier or host cell, and optional Pharmaceutically acceptable carrier or excipient.
In embodiments of the invention, possibly together with statins in described compositions.
In embodiments of the invention, described statins is selected from Seeley Wei Siting, atorvastatin, pungent cuts down Statin, Pitavastatin, Rosuvastatin, fluvastatin, lovastatin and pravastatin.
The invention still further relates to anti-PCSK9 antibody or its antigen-binding portion thereof, the present invention of any one of first aspect present invention The nucleic acid molecules of any one, carrier, host cell or compositions are in preparation prevention or treat cardiovascular disease or imbalance, thrombosis The purposes of the medicine of occlusive disease or imbalance.
In one embodiment of the invention, wherein said cardiovascular disease or imbalance are selected from dyslipidemia, crown Atherosclerotic heart disease, acute myocardial infarction, asymptomatic Carotid is atherosis, apoplexy and periphery artery occlusion disease Sick.
In one embodiment of the invention, wherein said dyslipidemia cholesterol in the blood raises, sweet Oil three fat raises, low density lipoprotein, LDL (LDL) raises and high density lipoprotein (HDL) reduces.A concrete reality in the present invention Executing in scheme, described dyslipidemia refers to hypercholesterolemia.
In one embodiment of the invention, wherein said thrombosis or imbalance selected from pulmonary infarction and regard Nethike embrane central vein thromboembolism.
The invention still further relates to anti-PCSK9 antibody or its antigen-binding portion thereof, the present invention of any one of first aspect present invention The nucleic acid molecules of any one, carrier, host cell or compositions be the use of the medicine of lipoprotein levels in preparation reduces blood On the way.
In embodiments of the invention, described lipoprotein is selected from cholesterol, triglyceride and low density lipoprotein, LDL.
Another aspect of the invention relates to the anti-PCSK9 antibody described in any one of the present invention or its antigen-binding portion thereof, basis Nucleic acid molecules, the carrier of the present invention, the host cell of the present invention, the conjugate of the present invention or the compositions of the present invention of invention Purposes in preparing following medicine:
The medicine that specificity is combined with PCSK9,
Block the medicine that PCSK9 with LDL-R is combined,
Improve the medicine of LDL-R level in cell surface LDL-R quantity or blood plasma,
Reduce the medicine of LDL or LDL-c level in blood plasma,
The medicine of LDL accumulation in suppression blood plasma,
The medicine of the LDL-R degraded that suppression PCSK9 is mediated, or
Improve the medicine of the metaboilic level of cholesterol and/or TG entrained by LDL.
Another aspect of the invention relate to a kind of in vivo or external method, arbitrary including the present invention using effective dose Anti-PCSK9 antibody described in Xiang or its antigen-binding portion thereof, the nucleic acid molecules of the present invention, the carrier of the present invention, the place of the present invention The step of the compositions of chief cell, the conjugate of the present invention or the present invention, described method is selected from as follows:
The method that specificity is combined with PCSK9,
Block PCSK9 Yu LDL-R associated methods,
Improve the method for LDL-R level in cell surface LDL-R quantity or blood plasma,
Reduce the method for LDL or LDL-c level in blood plasma,
The method of LDL accumulation in suppression blood plasma,
The method of the LDL-R degraded that suppression PCSK9 is mediated, or
Improve the method for the metaboilic level of cholesterol and/or TG entrained by LDL.
The invention still further relates to prevention or treatment cardiovascular disease or the method for imbalance, thrombosis or imbalance, institute The method of stating includes to experimenter in need prevention or the anti-PCSK9 antibody of any one of first aspect present invention of therapeutically effective amount Or its antigen-binding portion thereof, the nucleic acid molecules of any one of the present invention, carrier, host cell or the step of compositions.
In one embodiment of the invention, wherein said cardiovascular disease or imbalance are selected from dyslipidemia, crown Atherosclerotic heart disease, acute myocardial infarction, asymptomatic Carotid is atherosis, apoplexy and periphery artery occlusion disease Sick.
In one embodiment of the invention, wherein said dyslipidemia cholesterol in the blood raises, sweet Oil three fat raises, low density lipoprotein, LDL (LDL) raises and high density lipoprotein (HDL) reduces.A concrete reality in the present invention Executing in scheme, described dyslipidemia refers to hypercholesterolemia.
In one embodiment of the invention, wherein said thrombosis or imbalance selected from pulmonary infarction and regard Nethike embrane central vein thromboembolism.
The invention still further relates to reduce the method for lipoprotein levels in blood, described method includes preventing to experimenter in need Or the anti-PCSK9 antibody of any one of first aspect present invention of therapeutically effective amount or its antigen-binding portion thereof, any one of the present invention The step of nucleic acid molecules, carrier, host cell or compositions.
In embodiments of the invention, described lipoprotein is selected from cholesterol, triglyceride and low density lipoprotein, LDL.
The present invention obtains high affine from the Phage Antibody Library of phage antibody library and low capacity rite-directed mutagenesis The anti-PCSK9 antibody of power.It is demonstrated experimentally that the anti-PCSK9 antibody of the present invention is compared with existing antibody, there is higher affinity With can more effectively reduce LDL-c in hyperlipemia rhesus monkeys, have a good application prospect.
Hereinafter the present invention is described further:
In the present invention, unless otherwise stated, Science and Technology noun used herein has art technology The implication that personnel are generally understood that.Further, protein used herein and nucleic acid chemistry, molecular biology, cell and tissue Cultivation, microbiology, immunology relational language and laboratory operation step be corresponding field in widely used term and often Rule step.Meanwhile, in order to be more fully understood that the present invention, provide below definition and the explanation of relational language.
In the present invention, when mentioning PCSK9 albumen (Proprotein convertase subtilisin/kexin Type 9) aminoacid sequence time, it includes total length (such as people source NP_777596.21, the Mus source NP_ of PCSK9 albumen 705793.1 or monkey source NP_001106130.1)；Also include its fusion protein, such as with the Fc protein fragments of mice or human IgG (mFc or hFc) carries out the fragment merged.But, it will be appreciated by those skilled in the art that in the aminoacid sequence of PCSK9 albumen, can Naturally-produced or be artificially introduced sudden change or variation (include but not limited to displacement, lack and/or add), and do not affect its biology Function.
In the present invention, term EC50Refer to half-maximal effect concentration (concentration for 50%of maximal Effect), refer to cause the concentration of 50% ceiling effect.
In the present invention, term " antibody " refers to that generally (every pair has " gently " (L) chain to identical polypeptide chain by two With " weight " (H) chain) immunoglobulin molecules that forms.Light chain of antibody can be categorized as κ and lambda light chain.Heavy chain can be categorized as μ, δ, γ, α or ε, and respectively the isotype of antibody is defined as IgM, IgD, IgG, IgA and IgE.In light chain and heavy chain, can Becoming district and constant region to be connected by about 12 or more amino acid whose " J " district, heavy chain also comprises about 3 or more amino " D " district of acid.Each heavy chain is by variable region of heavy chain (VH) and CH (CH) composition.CH is by 3 domains (CH1、CH2 and CH3) composition.Each light chain is by variable region of light chain (VL) and constant region of light chain (CL) composition.Constant region of light chain is by one Domain CLComposition.The constant region of antibody can mediated immunity globulin and host tissue or the factor, including immune various The combination of first component (C1q) of cell (such as, effector lymphocyte) and classical complement system.VHAnd VLDistrict also can be subdivided into tool There is denatured region (referred to as complementary determining region (CDR)), be interspersed with the more conservative region being referred to as framework region (FR).Respectively VHAnd VLBy in the following order: arrange from amino terminal to carboxyl terminal 3 of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 CDR and 4 FR compositions.Variable region (the V of each heavy chain/light chain pairHAnd VL) form antibody combining site respectively.Aminoacid is to each district Kabat Sequences of Proteins of Immunological Interest is followed in the distribution of territory or domain (National Institutes of Health, Bethesda, Md. (1987and1991)), or Chothia&Lesk (1987) J.Mol.Biol.196:901-917；The definition of Chothia et al. (1989) Nature 342:878-883.Term " antibody " is not limited by the method for any specific generation antibody.Such as, it includes, especially, and recombinant antibodies, monoclonal antibody And polyclonal antibody.Antibody can be the antibody of different isotype, and such as, (such as, IgG1, IgG2, IgG3 or IgG4 are sub-for IgG Type), IgA1, IgA2, IgD, IgE or IgM antibody.
In the present invention, " antigen-binding portion thereof " of term antibody refers to one or more parts of full length antibody, described Part keeps the ability of same antigen (such as, PCSK9) that binding antibody is combined, with special to antigen of complete antibody competition Property combine.Generally see, Fundamental Immunology, Ch.7 (Paul, W., ed., second edition, Raven Press, N.Y. (1989), it passes through with it to quote to integrate with herein, for all purposes in full.Recombinant DNA technology can be passed through or pass through The enzymatic of complete antibody or chemical disruption produce antigen-binding portion thereof.In some cases, antigen-binding portion thereof include Fab, Fab'、F(ab')2、Fd, Fv, dAb and complementary determining region (CDR) fragment, single-chain antibody (such as, scFv), chimeric antibody, dual anti- Body (diabody) and such polypeptide, it comprises and be enough to give at least of the antibody of polypeptid specificity antigen binding capacity Point.
In the present invention, term " Fd fragment " means by VHAnd CHThe antibody fragment of 1 domain composition；Term " Fv fragment " Mean by the V of the single armed of antibodyLAnd VHThe antibody fragment of domain composition；Term " dAb fragment " means by VHDomain composition Antibody fragment (Ward et al., Nature 341:544-546 (1989))；Term " Fab fragment " means by VL、VH、CLAnd CH1 knot The antibody fragment of structure territory composition；Term " F (ab')2Fragment " mean to comprise two Fab connected by the disulphide bridges on hinge region The antibody fragment of fragment.
In some cases, the antigen-binding portion thereof of antibody is single-chain antibody (such as, scFv), wherein VLAnd VHDomain By can be produced as single polypeptide chain connector match formed monovalent molecule (see, e.g., Bird et al., Science 242:423-426 (1988) and Huston et al., Proc.Natl.Acad.Sci.USA 85:5879-5883 (1988)).This type of scFv molecule can have general structure: NH2-VL-joint-VH-COOH or NH2-VH-joint-VL- COOH.Suitably prior art joint (connection peptides) is made up of the GGGGS aminoacid sequence repeated or its variant.Such as, can make With having aminoacid sequence (GGGGS)4Joint, but be used as its variant (Holliger et al. (1993), Proc.Natl.Acad.Sci.USA90:6444-6448).Can be used for other joints of the present invention by Alfthan et al. (1995), Protein Eng.8:725-731, Choi et al. (2001), Eur.J.Immunol.31:94-106, Hu et al. (1996), Cancer Res.56:3055-3061, Kipriyanov et al. (1999), J.Mol.Biol.293:41-56 and Roovers et al. (2001), Cancer Immunol. describe.In embodiments of the invention, the sequence of described connection peptides is (GGGGS)3。
In some cases, antibody is bi-specific antibody, and it can respectively with two kinds of antigens or epitope combine, its Including the light chain of antibody, heavy chain or its antigen-binding portion thereof of specific binding first antigen, and specific binding second resists The light chain of former antibody, heavy chain or its antigen-binding portion thereof.In embodiments of the invention, described bi-specific antibody is tied Close antibody or its antigen knot that the light chain of the antibody of the first antigen, heavy chain or its antigen-binding portion thereof can be any one of the present invention Closing part, the light chain of antibody, heavy chain or its antigen-binding portion thereof of described specific binding second antigen can be that other resists PCSK9 antibody or its antigen-binding portion thereof or for the antibody of other antigen or its antigen-binding portion thereof.
In some cases, antibody is double antibody, i.e. bivalent antibody, wherein VHAnd VLDomain is table on single polypeptide chain Reach, but use the shortest connector thus not between two domains of same chain match, thus force domain with Another chain complementary domain pairing and produce two antigen-binding sites (see, e.g., Holliger P. et al., Proc.Natl.Acad.Sci.USA 90:6444-6448 (1993), and Poljak R.J. et al., Structure 2:1121- 1123(1994))。
Routine techniques well known by persons skilled in the art (such as, recombinant DNA technology or enzymatic or chemical disruption can be used Method) from the antigen-binding portion thereof (such as, above-mentioned antibody fragment) of given antibody (such as monoclonal antibody 2E12) acquisition antibody, And by with for complete antibody in the way of identical mode with regard to the antigen-binding portion thereof of specificity screening antibody.
In the present invention, described antigen-binding portion thereof includes single-chain antibody (scFv), chimeric antibody, double antibody, scFv- Fc bivalent molecule, dAb and complementary determining region (CDR) fragment, Fab fragment, Fd fragment, Fab' fragment, Fv and F (ab')2Fragment.
In the present invention, described IgG1 CH includes various allotype, as G1m (f), G1m (z), G1m (z, A) or G1m (z, a, x).In embodiments of the invention, described IgG1 CH is G1m (f) type.
In the present invention, described κ constant region of light chain includes various allotype, such as Km1, Km1, and 2 or Km3.In the present invention Embodiment in, described κ constant region of light chain is Km3 type.
In the present invention, described lambda light chain constant region includes various allotype, such as λ I, λ II, λ III, λ VI.In the present invention Embodiment in, described lambda light chain constant region is λ II type.
The antibody nucleic acids molecule that the present invention relates to can also utilize traditional genetic engineering recombinant technique or chemosynthesis side Method obtains.On the one hand, the sequence of the antibody nucleic acids molecule that the present invention relates to contains the variable region of heavy chain of anti-PCSK9 antibody or anti- The partial nucleic acid sequence of body molecule.On the other hand, the sequence of the antibody nucleic acids molecule that the present invention relates to also includes anti-PCSK9 antibody Variable region of light chain or the partial nucleic acid sequence of antibody molecule.On the other hand, the sequence of the antibody nucleic acids molecule that the present invention relates to Also include the CDR sequence of heavy chain or variable region of light chain.Complementary determining region (complementary determinant region, CDR) be the position being combined with epitope, the CDR sequence in the present invention by IMGT/V-QUEST (http: // Imgt.cines.fr/textes/vquest/) it is determined.But the CDR sequence that different division methods obtains is the most not With.
The present invention relates to the recombinant expression carrier containing described nucleic acid molecules, the host being directed to convert these molecules is thin Born of the same parents.And, the invention still further relates to utilize the host cell containing described nucleic acid molecules cultivate under given conditions and separate To the method inventing described antibody.
Antibody amino acids sequence
Antibody 8D8F heavy chain variable amino acid sequence is SEQ ID NO:15 respectively.Antibody 8D8F variable region of light chain amino Acid sequence is SEQ ID NO:16.
The aminoacid sequence of the heavy chain of antibody 8D8F and the CDR of variable region of light chain is defined below:
The aminoacid sequence of CDR1, CDR2 and CDR3 of antibody 8D8F heavy chain is respectively SEQ ID NO:1-3.
The aminoacid sequence of CDR1, CDR2 and CDR3 of antibody 8D8F light chain is respectively SEQ ID NO:4-6.
The aminoacid sequence of the heavy chain of antibody 8D8F and the FR of variable region of light chain is defined below:
The sequence of antibody 8D8F variable region of heavy chain FR1, FR2, FR3, FR4 is respectively SEQ ID NO:7-10.Light chain variable The sequence of district FR1, FR2, FR3, FR4 is respectively SEQ ID NO:11-14.
On the other hand, the heavy chain of anti-PCSK9 antibody or the aminoacid sequence of variable region of light chain FR are probably at SEQ ID NO:7-10,11-14 upper appearance one or more amino acid whose sudden changes, increase or lack.Preferably, suddenly change, add or lack The aminoacid lost is less than 3 aminoacid.It is further preferred that the aminoacid suddenling change, add or lacking is less than 2 aminoacid. Most preferably, the aminoacid suddenling change, add or lacking is less than 1 aminoacid.
Variant after the aminoacid of above-mentioned antibody or CDR region or framework region is undergone mutation, adds or lacked still is protected Stay the ability of specific binding people PCSK9.The present invention also comprises the variant of such antigen-binding portion thereof.
The monoclonal antibody variant of the present invention can be obtained by traditional gene engineering method.Those skilled in the art is complete Know the method utilizing nucleic acid mutation transformation DNA molecular.It addition, the nucleic acid molecules of encoding heavy chain and light chain variant can also lead to Cross chemosynthesis to obtain.
In the present invention, the algorithm for determining sequence iden and sequence similarity percent be such as BLAST and BLAST 2.0 algorithm, they be respectively described at (1977) Nucl.Acid.Res.25:3389-3402 such as Altschul and Altschul etc. (1990) J.Mol.Biol.215:403-410.Use such as described in document or default parameters, BLAST With the amino acid sequence identity percent that BLAST 2.0 is determined for the present invention.The software performing BLAST analysis is permissible By National Biotechnology, information centre is for the public to obtain.
In the present invention, the aminoacid sequence of the described sequence iden having at least 70% with aminoacid sequence include with The peptide sequence that described aminoacid sequence is the most same, such as when using methods described herein (for example with canonical parameter BLAST analyze) time, compared with peptide sequence of the present invention containing at least 70% sequence iden, preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or Those sequences of higher sequence iden.
In the present invention, term " carrier " refers to and can be inserted and make egg by the polynucleotide encoding certain albumen A kind of nucleic acid vehicle that BAIHUO must be expressed.Carrier by converting, can be transduceed or transfection host cell so that it is the heredity carried Substance element is expressed at host cell inner expression.For example, carrier includes: plasmid；Phasmid；Coemid；Manually Chromosome such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or the artificial chromosome (PAC) in P1 source；Phagocytosis Body such as bacteriophage lambda or M13 phage and animal virus etc..Animal virus kind as carrier has retrovirus (to include Slow virus), adenovirus, adeno-associated virus, herpesvirus (such as herpes simplex virus), poxvirus, baculovirus, papillomatosis Poison, papova viruses (such as SV40).The element that a kind of carrier may be expressed containing various control, including promoter sequence Row, transcriptional initiation sequence, enhancer sequence, selection element and reporter gene.It addition, carrier also can contain replication origin. Carrier is it is also possible to include and assist it to enter the composition of cell, such as virion, liposome or protein coat, but the most only There are these materials.
In the present invention, term " host cell " refers to import the cell of carrier, including following many cell types, as The prokaryotic cells such as escherichia coli or hay bacterium, such as the fungal cell such as yeast cells or aspergillosis, such as elder brothers such as S2 drosophila cell or Sf9 Worm cell, or such as fibroblast, Chinese hamster ovary celI, COS cell, NSO cell, HeLa cell, bhk cell, HEK 293 cell Or the zooblast of people's cell.
The antibody fragment of the present invention can utilize the complete antibody molecule of hydrolysis obtain (see Morimoto et al., J.Biochem.Biophys.Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)).It addition, these antibody fragments can also directly by recombinant host cell produce (reviewed in Hudson, Curr.Opin.Immunol.11:548-557 (1999)；Little et al., Immunol.Today, 21:364-370 (2000)).Such as, Fab' fragment can directly obtain from E.coli cell or chemistry Rhizoma Nelumbinis connection forms F (ab') 2 fragment (Carter et al., Bio/Technology, 10:163-167 (1992)).For another example, F (ab')2Fragment can use leucine Slide fastener GCN4 connects acquisition.It addition, Fv, Fab or F (ab')2Fragment can also be directly direct from recombinant host cell culture fluid Isolated.Those of ordinary skill in the art has full knowledge that other technology preparing antibody fragment.
In the present invention, " specific binding " refers to two intermolecular nonrandom association reactions, such as antibody and generation Reaction between the antigen of this antibody.Herein, it is detection in conjunction with the antibody of the first antigen to the binding affinity of the second antigen Less than or the most weak.In some embodiments, certain antigen-specific antibodies refers to affinity (KD)≤10-5M is (such as 10- 6M、10-7M、10-8M、10-9M、10-10M etc.) combine this antigen, wherein KDRefer to the ratio (koff/ of dissociation yield and combination rate Kon), it can use method familiar to the person skilled in the art to be measured.
In the present invention, term " KD" referring to the Dissociation equilibrium constant of specific antibodies-AI, it is used for describing Binding affinity between antibody and antigen.Equilibrium dissociation constant is the least, and antibody-antigen binding is the tightst, antibody and antigen it Between affinity the highest.Generally, antibody is with less than about 10-5M, the most about 10-6M、10-7M、10-8M、10-9M or 10-10Dissociation equilibrium constant (the K of M or lessD) conjugated antigen.It is, for example possible to use surface plasma body resonant vibration art (SPR) exists BIACORE instrument measures KD。
In embodiments of the invention, the anti-PCSK9 antibody of the present invention can specifically be tied with people's PCSK9 albumen Close.
In the present invention, term " effective dose " refers to the amount that be enough to obtain or at least partly obtain intended effect.Such as, Prevention condition effective amount refers to, it is sufficient to prevention, stops, or postpones the amount of the generation of disease；Treatment condition effective amount refers to, it is sufficient to Cure or at least partly stop the disease of the patient having suffered from disease and the amount of its complication.Measure such effective dose to exist completely Within the limit of power of those skilled in the art.Such as, amount effective for therapeutic use will depend upon which disease to be treated Severity, the immune overall status of patient oneself, the ordinary circumstance such as age of patient, body weight and sex, medicine Method of application, and the other treatment simultaneously used etc..
In embodiments of the invention, the anti-PCSK9 antibody of the present invention can suppress the combination of huPCSK9 Yu EGFA.
In the present invention, described blood includes whole blood, serum, blood plasma etc., the process of various blood samples and preparation method Well known in the art.
In the present invention, described hypercholesterolemia includes familial hypercholesterolemia and non-familial hypercholesterolemia Disease.
In the present invention, described dyslipidemia refers to compared with normal population, cholesterol in serum, triglyceride, low One or more in density lipoprotein raise, and/or the high density lipoprotein in serum reduces.
In the present invention, common usage is deferred in 20 kinds of conventional amino acid and its abbreviation.See Immunology-A Synthesis (second edition, E.S.Golub and D.R.Gren, Eds., Sinauer Associates, Sunderland, Mass. ), (1991) it is integrated with herein by quoting.
In embodiments of the invention, phage antibody library technique is used to screen a strain anti-PCSK9 monoclonal antibody. With existing anti-PCSK9 antibody is compared, the affinity of this strain antibody is higher, more can effectively reduce in hyperlipidemia monkey body LDL-c level, it is therefore expected that they will have a more preferable curative effect, and can reduce the effective dose of medicine, thus drop further The toxic and side effects of low medicine.And, the antibody of high-affinity has in terms of the stability and druggability of high concentration liquid preparation Certain advantage.
Accompanying drawing explanation
Fig. 1: the monoclonal phage ELISA relative affinity identifying phage-Abs.
The most often group bar diagram is followed successively by test antibodies, positive control (antibody mil66) and negative control from left to right (BSA)。
Fig. 2: the gradient dilution phage ELISA relative affinity identifying phage-Abs.
Fig. 3: pTGS-1 plasmid map.
(wherein DNA marker is Marker III to Fig. 4: PCR amplification 8D-CDR1,2M-Vkappa DNA fragmentation total length (TIANGEN, MD103)), total length is about 763bp.
Fig. 5: the gradient dilution phage ELISA relative affinity identifying phage-Abs.
The Binding experiment of Fig. 6: Mil66 and 8D8F Yu PCSK9.
Fig. 7: Mil66 and 8D8F Yu PCSK9 Competitive assays experiment.
Fig. 8: Mil66 and the 8D8F activity of cell biology that LDL is absorbed.
The regulation effect to LDL-c of Fig. 9: Mil66, the Mil68-4 antibody.
The regulation effect (decline percentage ratio) to LDL-c of Figure 10: Mil66, the Mil68-4 antibody.
The regulation effect to TC of Figure 11: Mil66, the Mil68-4 antibody.
The regulation effect (decline percentage ratio) to TC of Figure 12: Mil66, the Mil68-4 antibody.
Detailed description of the invention
Below in conjunction with embodiment, embodiment of the present invention are described in detail.Those skilled in the art will manage Solving, the following examples are merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.In embodiment unreceipted specifically Technology or condition person, (such as write with reference to J. Pehanorm Brooker etc., Huang according to the technology described by the document in this area or condition " the Molecular Cloning: A Laboratory guide " that training hall etc. is translated, the third edition, Science Press) or carry out according to product description.Agents useful for same Or instrument unreceipted production firm person, for buying, by market, the conventional products obtained.
Embodiment 1: the biopanning of anti-huPCSK9 single-chain antibody
Being antigen coated immunity pipe with huPCSK9-his (NP_777596.2), antigen coated amount is 5 μ g/ pipes, and 4 DEG C are coated Overnight.Defatted milk powder/the PBST using 4% closes off immunity pipe and naive phage antibody library (laboratory is prepared oneself), Close 1h for 37 DEG C.Carrying out antibody antigen combination in phage antibody library addition immunity pipe after closing, the input amount of phage is about It is 1012, 37 DEG C of reaction 1h.PBS (T) washes away unconjugated phage, and the HCL-Glycine antibody elution of 0.1M, with 1.5M's Tris-HCL (pH8.8) neutralizes the phage eluted.
Take in 550 μ l and after phage-infect about 10mL grow to the TG1 bacterium solution of exponential phase, 37 DEG C of standings 30min.Add the 9mL 2YT culture medium containing ammonia benzyl.Taking out appropriate bacterium solution bed board and measure cfu, 37 DEG C of remaining antibacterial cultivates 3h, expands Large volume, to 50mL, adds 2mL helper virus VCSM13 (pfu=1 × 1013), 30 DEG C of shaken cultivation are overnight.On reclaiming next day Clearly, conventional PEG precipitation, gained secondary phage antibody library can carry out the screening of next round.
Embodiment 2: the screening of anti-huPCSK9 single-chain antibody positive colony
The picking monoclonal bacterium colony that the separation of output is good after three-wheel screens, inoculates 1mL2YTAG In (Ampicilline:100 μ g/ml, Glucose:2%) culture medium, 37 DEG C, 220rpm overnight incubation.Within second day, it is forwarded to new 96 hole depth orifice plates, cultivate to its exponential phase, every hole adds about 1010Helper phage VCSM13.37 DEG C of static infection After 30min, 4000rpm, 4 DEG C of centrifugal 15min, supernatant discarded, thalline with 2YTAK (Ampicilline:100 μ g/ml, Kanamycin:70 μ g/ml) resuspended precipitation, 28 DEG C, 220rpm overnight incubation.Draw the phage supernatant after amplification to carry out ELISA identifies (seeing Fig. 1).The positive colony obtained is checked order, obtains 4 kinds of different antibody sequences altogether.
Embodiment 3:phage ELISA measures the affinity of anti-huPCSK9 single-chain antibody
The clone obtained in embodiment 2 is carried out displaying and the purification of monoclonal phage, carries out phage gradient dilution The affinity of ELISA experimental identification phage-abs.
Being coated huPCSK9 with the phosphate buffer of pH7.4,4 DEG C are coated overnight.PBST washs three times, 4%milk- PBST 37 DEG C closes 1h.Being diluted in proportion in the defatted milk powder of 4% by monoclonal phage after purification, every hole adds 100 μ Sample after l dilution, RT stands 1h.Elisa plate is washed, by the HRP-anti-M13 after 4% defatted milk powder dilution with PBST Monoclonal antibody adds in elisa plate, and room temperature places 1h.TMB colour reagent box develops the color, color development at room temperature 5min.Use 10%H2SO4 Color development stopping, 50 μ l/ holes.Microplate reader 450nm Single wavelength measures optical density value.Result shows the phagocytosis that several strains filtered out are different Body antibody all can be combined with huPCSK9, and the affinity of 8D clones (seeing Fig. 2) apparently higher than other.
Embodiment 4: the anti-huPCSK9 single-chain antibody 8D filtered out is carried out external affinity maturation
PComb3 carrier (is protected by the method using series of genes clone purchased from China's plasmid vector strain cell pnca gene Center, Tibetan) transform, it is allowed to be applicable to the structure of phage antibody library and expression: use the side of series of genes clone Method removes the light chain cloning region region in pComb3, simultaneously to heavy chain cloning Region transforms, and removes the CH1 part in heavy chain cloning region, then by PCR method at many grams Grand site adds NcoI and NotI restriction enzyme site, makes it possible to clone and expresses ScFv single-chain antibody.Improved carrier is ordered Entitled pTGS-1, its plasmid map is as it is shown on figure 3, and build rite-directed mutagenesis antibody to the VH part of 8D based on pTGS-1 Storehouse, carries out antibody in vitro affinity maturation.
The structure of 1.8D-CDR1,2M antibody library
Respectively with 8D as template, utilize rite-directed mutagenesis PCR reaction amplification heavy chain 8D-CDR1,2 district's gene and 8D- CDR3-Vkappa district gene (wherein Vkappa refers to the variable region of light chain).QIAgene glue reclaims test kit and reclaims PCR sheet Section.The DNA fragmentation that above-mentioned two parts PCR reaction obtains is carried out Overlap PCR, and 8D-CDR1,2M-Vkappa are complete in amplification Long.Reaction condition: 95 DEG C of 30s, [95 DEG C of 10s, 60 DEG C of 30s, 72 DEG C of 50s] 3cycle, [95 DEG C of 10s, 72 DEG C of 50s] 25cycles, 72 DEG C of 2min, 4 DEG C of preservations.QIAgene glue reclaims test kit and reclaims PCR fragment (seeing Fig. 4).
Respectively plasmid pTGS-1 and 8D-CDR1,2M-Vkappa PCR total length are carried out double with NcoI-HF and NotI Enzyme action.Plasmid pTGS-1, through 1.5% agarose gel electrophoresis, cuts glue and reclaims.PCR fragment uses QIAgene PCR Purification kit test kit reclaims.PCR fragment after recovery and pTGS-1 plasmid 8:1 ratio warp in molar ratio T4DNA ligase room temperature connects overnight.Junction fragment electric shocking method is converted to TG1 competence.Use SOC culture medium 37 DEG C training Support 1h recovery antibacterial.Take certain bacterium solution, be coated with flat board, calculate storage capacity.Remaining bacterium solution 4000rpm, centrifugal 15min under room temperature.Abandon Supernatant, precipitation is coated on 2～3 2YTCG massive plates, is inverted overnight incubation for 37 DEG C.
Antibody library storage capacity is about 104, from this antibody library, random 10 clones of picking carry out sequence analysis, and accuracy is 70%.
2. the biopanning of phage antibody library and the screening of positive colony
By the antibody library of above-mentioned structure, carry out phage displaying, purification and precipitation.Then the anti-huPCSK9 of elutriation from this storehouse Single-chain antibody.The biopanning method of phage antibody library is with embodiment 1, and (chlorine is mould simply Amp resistance to be replaced to Cm resistance Element: Chloramphenicol), helper virus is replaced with M13KO7 by VCSM13.Anti-huPCSK9 single-chain antibody positive colony Amp resistance, with embodiment 2, is simply replaced to Cm resistance by screening technique, and helper virus is replaced with M13KO7 by VCSM13.Screening To 1 strain positive monoclonal, its expressed sequence anti-huPCSK9 antibody unlike the prior art, named 8D8F.
3.phage ELISA measures the affinity of 8D8F single-chain antibody
8D8F is carried out displaying and the purification of monoclonal pahge, phage gradient dilution ELISA experimental identification phage- The affinity of abs, method is with embodiment 3.Result shows, the affinity of 8D8F is slightly below 8D (seeing Fig. 5).
Aminoacid and the nucleotide sequence of 8D8F antibody are as follows:
8D8F VH
EVQLVESGGGLVQPGGSLRLSCAASGFAFGGYAMNWVRQAPGKGLDWVSTISGSGGSTNYADSVKGRFIISRDSSKH TLYLQMNSLRAEDTAVYYCAKDSNWGNFDLWGRGTLVTVSS(SEQ ID NO:15)
The nucleotide sequence of coding 8D8F VH
GAGGTGCAGCTGGTGGAGAGCGGTGGTGGCCTGGTGCAGCCGGGCGGCAGCCTGCGTCTGAGCTGCGCCGCCAGCGG CTTCGCTTTCGGCGGTTACGCTATGAACTGGGTGCGCCAGGCCCCTGGCAAGGGCCTGGACTGGGTGAGCACCATTA GCGGTAGCGGTGGCAGCACCAACTACGCCGACAGCGTGAAGGGCCGTTTCATCATCAGCCGCGACAGCAGCAAGCAC ACCCTGTACCTGCAGATGAACAGCCTGCGTGCCGAGGACACCGCCGTGTACTACTGCGCCAAGGACAGCAACTGGGG CAACTTCGACCTGTGGGGCCGCGGCACCCTGGTGACCGTGAGCAGC(SEQ ID NO:17)
8D8F VL
DIVMTQSPDSLAVSLGERATINCKSSESVMYRRNARNFLGWYQQKPGQPPNLLIYWASTRESGVPDRFSGSGSGTDF TLTISSLQAEDVAVYYCQQYYTHPYTFGQGTKLEIK(SEQ ID NO:16)
The nucleotide sequence of coding 8D8F VL
GACATCGTGATGACCCAGAGCCCGGACAGCCTGGCCGTGAGCCTGGGCGAGCGCGCCACCATCAACTGCAAGAGCAG CGAAAGCGTGATGTACCGTCGCAACGCTCGCAACTTCCTGGGCTGGTACCAGCAGAAGCCTGGCCAGCCTCCTAACC TGCTGATCTACTGGGCCAGCACCCGTGAGAGCGGCGTGCCTGACCGCTTCTCTGGTAGCGGCTCTGGCACCGACTTC ACCCTGACCATCAGCAGCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCAGCAGTACTACACCCACCCTTACAC CTTCGGCCAGGGCACCAAGCTGGAGATCAAG(SEQ ID NO:18)
In above sequence, the part of band underscore is CDR region sequence.
Wherein:
8D8F VH CDR1:SGFAFGGYAMN (SEQ ID NO:1)
8D8F VH CDR2:TISGSGGSTN (SEQ ID NO:2)
8D8F VH CDR3:AKDSNWGNFDL (SEQ ID NO:3)
8D8F VL CDR1:KSSESVMYRRNARNFLG (SEQ ID NO:4)
8D8F VL CDR2:WASTRESGVPDR (SEQ ID NO:5)
8D8F VL CDR3:QQYYTHPYT (SEQ ID NO:6)
VH FR1:EVQLVESGGGLVQPGGSLRLSCAA (SEQ ID NO:7)
VH FR2:WVRQAPGKGLDWVS (SEQ ID NO:8)
VH FR3:
YADSVKGRFIISRDSSKHTLYLQMNSLRAEDTAVYYC(SEQ ID NO:9)
VH FR4:WGRGTLVTVSS (SEQ ID NO:10)
VL FR1:DIVMTQSPDSLAVSLGERATINC (SEQ ID NO:11)
VL FR2:WYQQKPGQPPNLLIY (SEQ ID NO:12)
VL FR3:FSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO:13)
VL FR4:FGQGTKLEIK (SEQ ID NO:14)
The Function Identification of embodiment 5:8D8F antibody
Chain variable region gene and the heavy chain variable region gene of 8D8F are cloned into equipped with constant region of light chain and light chain constant District (CH is the constant region of human IgG1, its aminoacid sequence as shown in SEQ ID NO:19, nucleotide sequence such as SEQ Shown in ID NO:20；The constant region of constant region of light chain behaviour kappa, its aminoacid sequence as shown in SEQ ID NO:21, nucleoside Acid sequence is as shown in SEQ ID NO:22) on the pCDNA3.1 carrier of gene, transfection CHO-K1 cell (purchased from U.S. ATCC, CCL-61TM), carry out whole antibody secreting, expressing, through protein A purification, after super filter tube ultrafiltration, obtain whole antibody albumen.With Mil66 is that (identical with the Alirocumab sequence of Sai Nuofei/regeneration unit, the expression according to 8D8F antibody is pure for positive control Its expression and purification is obtained by change method；Its sequence can also be with reference to INN 9620_H, INN 9620_L.), obtain in the present invention Anti-huPCSK9 antibody 8D8F carry out functional verification.
1. the affinity of gradient dilution method detection whole antibody 8D8F and Mil66 and PCSK9
Being coated PCSK9 antigen with PBS, being coated concentration is 5 μ g/ml, and 4 DEG C are coated overnight.PBST washes elisa plate, totally three times. 1%BSA-PBST 37 DEG C closes 1h.The mol ratio of 8D8F and Mil66 and PCSK9 is from the beginning of 1:1, and three times of gradient dilutions, when dense When degree is to 41.2ng/ μ l, carry out two-fold dilution.Each sample does 11 gradient dilutions, and last hole is as negative control.By dilute The sample released adds in elisa plate, and 1h is hatched for 37 DEG C in 100 μ l/ holes.Wash three times, by HRP labelling with 200 μ l/ hole PBST Goat anti human IgG bis-is anti-to be diluted in 1%BSA-PBST by 1:2000, hatches 1h for 37 DEG C.TMB colour reagent box develops the color, 100 μ L/ hole, color development at room temperature 5min.Use 10%H2SO4Color development stopping, 50 μ l/ holes.Reading under the wavelength of 450nm.
Result such as Fig. 6 and table 1 below.
The EC that table 1:8D8F and Mil66 antibody are combined with PCSK950Value
Mil66 8D8F
EC<sub>50</sub>(ng/ml) 45.72 6.58
Result shows, 8D8F with Mil66 all can be combined with PCSK9.But the binding ability of 8D8F is apparently higher than Mil66, Its medium effective concentration EC50It is worth higher about 6.6 times than Mil66.
2.BIAcore measures the affinity of 8D8F whole antibody
Prize law is used to measure the affinity of whole antibody.By anti-human FCThe antibody coupling of section on the surface of chip CM5, point Do not dilute Mil66,8D8F antibody to 1 μ g/ml, it is ensured that about 100RU antibody is by the antibody capture of anti-human Fc.By huPCSK9-his A series of Concentraton gradient (200nm, 100nm, 50nm, 25nm, 12.5nm, 6.25nm) is set and flows through fixing phase surface, measure The affinity of antibody.Result is as shown in Table 2 below.
Table 2:Mil66 and 8D8F affinity of antibody constant measuring
Result shows, the K of 8D8F antibodyDIt is worth and improves about 7.7 times than Mil66.
3.8D8F whole antibody is tested with PCSK9 Competitive assays
Being coated EGFA-Fc with the carbonate buffer solution of pH9.6, being coated concentration is 2 μ g/ml, and 4 DEG C are coated overnight.PBST washes Washing three times, 1%BSA-PBST 37 DEG C closes 1h.Then 3 times of gradients are carried out to 10 μ g/ml dilute with 1%BSA-PBST dilution antibody Release, 10 dilution gradients are set altogether, in the sample diluted, then add the huPCSK9-his of isopyknic 8 μ g/ml, 37 DEG C hatch 1h.Then the mixture hatched is added in elisa plate, hatch 1h for 37 DEG C.Wash three times with 200 μ l/ hole PBST, will The little mouse-anti His bis-of HRP labelling is anti-to be diluted in 1%BSA-PBST by 1:3000, hatches 1h for 37 DEG C.TMB colour reagent box shows Color, 100 μ l/ holes, color development at room temperature 8min.Use 10%H2SO4Color development stopping, 50 μ l/ holes.450nm reading.Result such as Fig. 7 and following Table 3.
Table 3Mil66,8D8F and PCSK9 Competitive assays IC50Value
Mil66 8D8F
IC<sub>50</sub>(ng/ml) 15.43 30.9
Result shows, Mil66 and 8D8F all can suppress the combination of huPCSK9 Yu LDLR.
The activity of cell biology that LDL is absorbed by 4.Mil66,8D8F
Being inoculated on 96 orifice plates with the amount in 40000/hole by HepG2 cell with complete medium (DMEM), culture plate is put To 37 DEG C of cell culture incubator (5%CO2After cultivating 24h in), sucking-off supernatant, add the DMEM culture medium containing 1%FBS and continue to cultivate 16h.After anti-PCSK9 antibody (Mil66,8D8F) is diluted to 400 μ g/ml, 2 times of gradient dilutions, altogether 8 concentration of dilution, HuPCSK9-his is diluted to 800 μ g/ml, the antibody diluted and antigen simultaneously mix with the ratio of 1:1,37 DEG C of cell trainings Foster case is hatched 30 minutes, supernatant in sucking-off subsequently 96 orifice plate, adds the antigen-antibody mixture after hatching, is simultaneously introduced LDL, The final concentration of 10 μ g/ml of LDL, put supernatant sucking-off, PBS Tissue Culture Plate to 37 DEG C of cell culture incubators after cultivating 6 hours After cleaning twice, culture plate puts into fluorescence intensity in microwell plate detecting system, a length of 485nm of excitation light wave, launches light wave A length of 535nm.With antibody concentration as abscissa, Reversal of PCSK9effect on LDL uptake (%) sits for vertical Mark, matching quadruplex parameters curve calculates EC50Value.During sample detection, each plate adds Mil66 as comparison.
Result is as shown in Figure 8.
Result shows, Mil66,8D8F all can effectively stop the combination of huPCSK9-his Yu LDLR, thus reverse The inhibitory action that LDL is absorbed by huPCSK9-his.
Embodiment 6:Mil66, the 8D8F blood lipid regulation test of pesticide effectiveness to hyperlipemia Rhesus Macacus
8D8F is renamed as Mil68-4.Evaluate Mil66 and the Mil68-4 antibody blood to hyperlipemia Rhesus Macacus Fat regulation effect.
Selected 15 the hyperlipemia Rhesus Macacus of this test, body weight 7-15kg, the horizontal 1.1-1.7mmol/ of internal LDL-c L.Monkey is divided into 2 groups: Mil68-4 (1.2mg/kg) and MIL66 (1.2mg/kg) group, often 3 hyperlipemia Rhesus Macacus of group, single Secondary subcutaneous administrations.Gather blood sample the most respectively 2 times, within 1,3,5,7,14 days, respectively gather blood sample one the most upon administration Secondary, analyze for blood lipids index detection, it may be assumed that T-CHOL (TC), triglyceride (TG), low density lipoprotein, LDL (LDL-c), high density Lipoprotein (HDL-c).Animal before sampling operation, fasting 14-16h, do not anaesthetize, through forearm venous blood collection 0.5ml.Use one Roche Cobas C501 detects blood fat indices.
Experimental result is as shown in Fig. 9 to Figure 12.
Result shows, (1.2mg/kg is equivalent to clinical dosage 42mg/ people), MIL66 and Mil68-4 under Isodose After medicine single-dose, the action time to LDL-c is similar, and first day LDL-c is remarkably decreased upon administration, and downward trend It is continued for administration 5-7 days.Wherein Mil68-4 reduced 51.95%-49.45% at D5-D7 days, and Mil66 exists Within D5-D7 days, reduce 44.05%-40.87%.After D7 days, the drug action of Mil66 has weakened, and within D14 days, drops to 21.73%.And the drug action of Mil68-4 can continue to 14 days, within D14 days, LDL-c reduces by 44.34%.
As can be seen from the above results, Mil68-4 is substantially better than positive control to action intensity and the persistent period of LDL-c Mil66 (Fig. 9,10).After MIL66 and Mil68-4 single-dose, the regulation effect to hyperlipemia Rhesus Macacus TC is suitable.With baseline Value compares, and TC persistently reduced from first day to the 5th day.In terms of the data of the 1st, 3,5 days, MiL66 reduces the intensity of TC and is slightly better than Mil68-4, but Mil68-4 drug action can continue to the 14th day, and Mil66 drug action after the 7th day weakens (ginseng See Figure 11,12).After Mil66 and Mil68-4 medicine single-dose, HDL-c and TG be there are no obvious effect.
Although the detailed description of the invention of the present invention has obtained detailed description, it will be understood to those of skill in the art that.Root According to disclosed all teachings, those details can be carried out various amendment and replacement, these change all the guarantor of the present invention Within the scope of protecting.The four corner of the present invention is given by claims and any equivalent thereof.

Claims (21)

  1. The most anti-PCSK9 antibody or its antigen-binding portion thereof, wherein:
    The variable region of heavy chain of described anti-PCSK9 antibody comprises: aminoacid sequence is the CDR1-CDR3 of SEQ ID NO:1-3；
    And/or
    The variable region of light chain of described anti-PCSK9 antibody comprises: aminoacid sequence is the CDR1-CDR3 of SEQ ID NO:4-6.
  2. Anti-PCSK9 antibody the most according to claim 1 or its antigen-binding portion thereof, its variable region of heavy chain FR1, FR2, FR3, FR4 district comprises the sequence as shown in SEQ ID NO:7-10 respectively, or comprises the homogeneity with above-mentioned sequence more than 70%, Be greater than 75%, more than 80%, more than 85%, more than 90%, more than 95% or more than 99% sequence；
    And/or
    FR1, FR2, FR3, FR4 district, its variable region of light chain comprises the sequence as shown in SEQ ID NO:11-14, or bag respectively Containing the homogeneity with above-mentioned sequence more than 70%, it is greater than 75%, more than 80%, more than 85%, more than 90%, more than 95% Or the sequence more than 99%.
  3. 3. according to the anti-PCSK9 antibody described in any claim in claim 1-2 or its antigen-binding portion thereof, its heavy chain Constant region is selected from deriving from the IgG (such as IgG1, IgG2, IgG3 or IgG4) of people, IgM, IgE, IgD and IgA.
  4. 4. according to the anti-PCSK9 antibody of any claim in claim 1-3 or its antigen-binding portion thereof, its chain constant District is κ or λ deriving from people.
  5. 5. according to the anti-PCSK9 antibody of any claim in claim 1-4 or its antigen-binding portion thereof, its be whole antibody, Bi-specific antibody, scFv, Fab, Fab', F (ab')2Or Fv.
  6. 6. according to the anti-PCSK9 antibody described in any claim in claim 1 to 5 or its antigen-binding portion thereof, wherein, institute The anti-PCSK9 antibody stated is with less than about 10-5M, the most about 10-6M、10-7M、10-8M、10-9M or 10-10M or less KDIn conjunction with PCSK9 albumen.
  7. 7. a nucleic acid molecules, its anti-PCSK9 antibody containing coding any one of claim 1-6 or its antigen-binding portion thereof Or the sequence of its fragment；Preferably, described nucleic acid molecules has the nucleotide sequence shown in SEQ ID NO:17 and/or SEQ ID Nucleotide sequence shown in NO:18.
  8. 8. a carrier, it contains the nucleic acid molecules described in claim 7.
  9. 9. a host cell, it contains the nucleic acid molecules described in claim 7 or the carrier described in claim 8.
  10. 10. a conjugate, it includes antibody or its Fab and coupling moiety, and wherein, described antibody is right Requiring the anti-PCSK9 antibody described in any claim or its antigen-binding portion thereof in 1 to 6, described coupling moiety is for detecting Labelling；Preferably, described coupling moiety is radiosiotope, fluorescent material, luminescent substance, coloring matter or enzyme.
  11. 11. 1 kinds of test kits, it includes the anti-PCSK9 antibody described in any claim or its antigen knot in claim 1 to 6 Close part, or include the conjugate described in claim 10；
    Preferably, described test kit also includes second antibody, anti-PCSK9 antibody or its antigen-binding portion described in its specific recognition Point；Optionally, described second antibody also includes detectable labelling, such as radiosiotope, fluorescent material, luminescent substance, Coloring matter or enzyme.
  12. In 12. claim 1 to 6, anti-PCSK9 antibody described in any claim or its antigen-binding portion thereof or right are wanted Seek the purposes in preparing test kit of the conjugate described in 10, described test kit for detect PCSK9 existence in the sample or Its level.
  13. 13. 1 kinds of compositionss (such as pharmaceutical composition), it contains resisting described in any claim in claim 1-6 PCSK9 antibody or the nucleic acid molecules described in its antigen-binding portion thereof, claim 7, the carrier described in claim 8, right are wanted Ask the host cell described in 9 or the conjugate described in claim 10, and optional pharmaceutically acceptable carrier or tax Shape agent.
  14. 14. compositionss according to claim 13, it is possibly together with statins, is selected from Seeley Wei Siting, atropic Cut down the one in statin, simvastatin, Pitavastatin, Rosuvastatin, fluvastatin, lovastatin and pravastatin or many Kind.
  15. Anti-PCSK9 antibody described in any claim or its antigen-binding portion thereof, claim 7 in 15. claim 1-6 Carrier described in described nucleic acid molecules, claim 8, the host cell described in claim 9, the idol described in claim 10 Compositions described in connection thing or claim 13 or 14 is in preparation prevention or treats cardiovascular disease or imbalance, rhomboembolia type Purposes in the medicine of disease or imbalance.
  16. 16. purposes according to claim 15, wherein said cardiovascular disease or imbalance are selected from dyslipidemia, crown dynamic Arteries and veins hardening heart disease, acute myocardial infarction, asymptomatic Carotid is atherosis, apoplexy and Peripheral arterial occlusive disease.
  17. 17. purposes according to claim 16, wherein said dyslipidemia cholesterol in blood raises, glycerol Three fat raise, low density lipoprotein, LDL (LDL) raises and high density lipoprotein (HDL) reduces.
  18. 18. purposes according to claim 16, wherein said thrombosis or imbalance selected from pulmonary infarction and regard Nethike embrane central vein thromboembolism.
  19. In 19. claim 1-6 described in the anti-PCSK9 antibody of any claim or its antigen-binding portion thereof, claim 7 Nucleic acid molecules, the carrier described in claim 8, the host cell described in claim 9, the conjugate described in claim 10 Or the compositions described in claim 13 or 14 is the purposes in the medicine of lipoprotein levels in preparation reduces blood.
  20. In 20. claim 1-6 described in the anti-PCSK9 antibody of any claim or its antigen-binding portion thereof, claim 7 Nucleic acid molecules, the carrier described in claim 8, the host cell described in claim 9, the conjugate described in claim 10 Or the purposes that the compositions described in claim 13 or 14 is in preparing following medicine:
    The medicine that specificity is combined with PCSK9,
    Block the medicine that PCSK9 with LDL-R is combined,
    Improve the medicine of LDL-R level in cell surface LDL-R quantity or blood plasma,
    Reduce the medicine of LDL or LDL-c level in blood plasma,
    The medicine of LDL accumulation in suppression blood plasma,
    The medicine of the LDL-R degraded that suppression PCSK9 is mediated, or
    Improve the medicine of the metaboilic level of cholesterol and/or TG entrained by LDL.
  21. 21. 1 kinds in vivo or external method, including using the anti-of any claim in claim 1-6 of effective dose PCSK9 antibody or the nucleic acid molecules described in its antigen-binding portion thereof, claim 7, the carrier described in claim 8, right are wanted Ask the host cell described in 9, the conjugate described in claim 10 or the step of the compositions described in claim 13 or 14, Described method is selected from as follows:
    The method that specificity is combined with PCSK9,
    Block PCSK9 Yu LDL-R associated methods,
    Improve the method for LDL-R level in cell surface LDL-R quantity or blood plasma,
    Reduce the method for LDL or LDL-c level in blood plasma,
    The method of LDL accumulation in suppression blood plasma,
    The method of the LDL-R degraded that suppression PCSK9 is mediated, or
    Improve the method for the metaboilic level of cholesterol and/or TG entrained by LDL.
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WO2018113781A1 (en) * 2016-12-24 2018-06-28 信达生物制药(苏州)有限公司 Anti-pcsk9 antibody and application thereof
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WO2021052472A1 (en) * 2019-09-19 2021-03-25 信达生物制药(苏州)有限公司 Method for preventing or treating cholesterol-related diseases by using anti-pcsk9 antibody
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CN114438075A (en) * 2022-02-25 2022-05-06 刘博巽 Screening method and application of bacillus subtilis proprotease 9 targeted binding protein

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