CN106084058B - Anti-human PCSK9 monoclonal antibody - Google Patents
Anti-human PCSK9 monoclonal antibody Download PDFInfo
- Publication number
- CN106084058B CN106084058B CN201610540921.6A CN201610540921A CN106084058B CN 106084058 B CN106084058 B CN 106084058B CN 201610540921 A CN201610540921 A CN 201610540921A CN 106084058 B CN106084058 B CN 106084058B
- Authority
- CN
- China
- Prior art keywords
- antibody
- pcsk9
- antigen
- binding portion
- ldl
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- 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
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- 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
-
- 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
- C07K2317/565—Complementarity determining region [CDR]
-
- 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
- C07K2317/567—Framework region [FR]
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- 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)
Abstract
The invention belongs to antibody arts, and in particular to anti-human PCSK9 monoclonal antibody and the antibody are used to prepare the purposes of the lipoprotein levels reduced in blood, prevention or treatment cardiovascular disease or the drug of imbalance, thrombosis or imbalance.Anti-human PCSK9 monoclonal antibody affinity of the invention is higher, has a good application prospect.
Description
Technical field
The invention belongs to antibody arts, and in particular to anti-human PCSK9 monoclonal antibody and the antibody are used to prepare drop
Lipoprotein levels, prevention in low blood or treatment cardiovascular disease or the drug of imbalance, thrombosis or imbalance
Purposes.
Background technique
Proprotein convertase subtilisin/kexin9 (Proprotein Convertase Subtilisin/
Kexin type 9, PCSK9) it is the unique proprotein convertases for belonging to serine protease K subclass.The preceding albumen conversion
Enzyme (PCSK9) assignment of genes gene mapping is made of, code area is about in human chromosomal 1p32.3, overall length 29kb 12 exons
2kb, coding include the protein of 692 amino acid residues.PCSK9 is mainly by signal peptide, predomain, catalyst structure domain, and
Carboxy-terminal domains composition.PCSK9 precursor protein synthesizes a kind of soluble proenzyme, i.e. PCSK9 proenzyme (apo- in endoplasmic reticulum
PCSK9,74KD), autocatalysis division release occurs at apo-PCSK9 151-152 residue in endoplasmic reticulum or golgiosome
Propetide (14KD) forms maturation protein enzyme (60KD) and is secreted into blood.PCSK9 is mainly expressed in liver, small intestine, but only
It can be secreted into blood in the PCSK9 of liver expression.
Serum low-density LP level is to measure the main indicator of blood lipid level, and LDL raising is atherosclerotic
Cardiopathic Major Risk Factors can induce and promote the occurrence and development of atherosclerosis.It is numerous research shows that PCSK9 can be with
The low density lipoprotein cholesterol receptor (low-density lipoprotein receptor, LDL-R) of cell surface combines
And guidance is internalized by lysosomal degradation, inhibit it to be recycled to surface of hepatocytes, to weaken low in liver metabolism blood plasma
The ability of density lipoprotein-cholesterol (LDL-c).Epidemiological study discovery, PCSK9 gene different parts base mutation can be led
Two kinds of completely different biological effects are caused, one is the mutation of function acquisition type, another kind is Loss-of-function mutation.Function obtains
The type mutation of obtaining can enhance the ability of degradation liver cell LDLR, reduce so that the LDL-c in blood is removed, lead to high cholesterol
The generation of mass formed by blood stasis.Loss-of-function is mutated the normal function that can destroy PCSK9, causes surface of hepatocytes LDLR to increase, to make
It obtains LDL-c in blood and removes and increase.Studies have shown that low-level blood caused by this gene function defect because of PCSK9
LDL-c can significantly reduce the disease incidence of atherosclerosis and coronary heart disease.Genetics research is further discovered that in PCSK9 egg
In the white crowd lacked completely, their intracorporal low-density lipoprotein white levels are only 1/10th of general population, and this feelings
There is no have any impact to their health to condition.This explanation should be not present any by the function of Drug inhibition PCSK9
Side effect.Therefore, become the hot spot of recent researches by inhibiting PCSK9 to achieve the purpose that reduce LDL-c.
In the research and development of numerous PCSK9 inhibitor, anti-PCSK9 monoclonal antibody attracts attention.It is more large-scale at present international
Drugmaker is all directed to the monoclonal antibody medicine of huPCSK9, the wherein Evolocumab of Amgen and Sino phenanthrene/regeneration in active development
III clinical trial phase has been completed in the Alirocumab of member.III phase clinical data is shown, is compared with standard treatment, both antibody
61% LDL-c, 1 year 50% cardiovascular event of reduction can be reduced, long-term efficacy is had an optimistic view of more.
Human antibody is the Main way of therapeutic antibodies development, and the appearance of phage antibody library technique is anti-for full source of people
The preparation of body provides good technology platform.Phage antibody library technique uses PCR method amplification in vitro human antibody VH and VL
Portion gene, then will be on the random recombinant clone to Vector for Phage Display of VH and VL.It is presented on the antibody energy of phage surface
It is enough to interact in vitro with immobilised targeting antigen, by washing removal non-specific binding antibody repeatedly, then elute
And collecting is enriched with the bacteriophage of specificity with the bacteriophage of antigen binding, bacteriophage ehec infection again.Most
It is worked afterwards by DNA sequencing, obtains the antibody sequence with targeting antigen specific binding.Phage antibody library technique simulates body
The selection index system of interior immune system and the process of affinity matured antibody, need not move through hybridoma technology, even without by exempting from
Epidemic disease process can be obtained specificity and high-affinity various Antibody molecule fragments, substantially reduce the research and development time of antibody.
This field still lacks the anti-human PCSK9 human antibody with more high-affinity.
Summary of the invention
The present invention first rapidly filters out the monoclonal antibody of anti-huPCSK9 from naive phage antibody library, then answers
With building low capacity rite-directed mutagenesis fully synthetic phage antibody library method, to the weight of antibody on the basis of the antibody filtered out
Chain variable region CDR1,2,3 region mutations build the external affinity maturation that library carries out antibody, obtain the antibody of high-affinity, thus
Complete the present invention.
The first aspect of the present invention is related to anti-PCSK9 antibody or its antigen-binding portion thereof comprising selected from such as next group
CDR region:
The area heavy chain variable region CDR1, CDR2, CDR3 separately includes the sequence as shown in SEQ ID NO:1,2,3, and light chain can
Become the area area CDR1, CDR2, CDR3 and separately includes the sequence as shown in SEQ ID NO:4-6；
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, the anti-PCSK9 antibody or its antigen-binding portion thereof, in which:
The heavy chain variable region of the anti-PCSK9 antibody includes the CDR1- that amino acid sequence is SEQ ID NO:1-3
CDR3；
And/or
The light chain variable region of the anti-PCSK9 antibody includes the CDR1- that amino acid sequence is SEQ ID NO:4-6
CDR3。
In one embodiment of the invention, the antibody or heavy chain variable region FR1, FR2 of its antigen-binding portion thereof,
The area FR3, FR4 separately includes the sequence as shown in SEQ ID NO:7-10, or comprising being greater than with the identity of above-mentioned sequence
70%, it is greater than 75%, 80%, 85%, 90%, 95%, 99% sequence.
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 antibody or light chain variable region FR1, FR2 of its antigen-binding portion thereof,
The area FR3, FR4 separately includes the sequence as shown in SEQ ID NO:11-14, or comprising being greater than with the identity of above-mentioned sequence
70%, it is greater than 75%, 80%, 85%, 90%, 95%, 99% sequence.
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 heavy chain variable region of the antibody or its antigen-binding portion thereof
Column are as shown in SEQ ID NO:15.Underscore part is CDR sequence.
EVQLVESGGGLVQPGGSLRLSCAASGFAFGGYAMNWVRQAPGKGLDWVSTISGSGGSTNYADSVKGRF
IISRDSSKHTLYLQMNSLRAEDTAVYYCAKDSNWGNFDLWGRGTLVTVSS(SEQ ID NO:15)
In one embodiment of the invention, the sequence of the light chain variable region of the antibody or its antigen-binding portion thereof
Column are as shown in SEQ ID NO:16.Underscore part is CDR sequence.
DIVMTQSPDSLAVSLGERATINCKSSESVMYRRNARNFLGWYQQKPGQPPNLLIYWASTRESGVPDRF
SGSGSGTDFTLTISSLQAEDVAVYYCQQYYTHPYTFGQGTKLEIK(SEQ ID NO:16)
In one embodiment of the invention, the antibody or the heavy chain constant region of its antigen-binding portion thereof derive from
People.
In one embodiment of the invention, the antibody or the heavy chain constant region of its antigen-binding portion thereof are selected from source
In IgG, IgM, IgE, IgD and IgA of people.
In one embodiment of the invention, the antibody or the constant region of light chain of its antigen-binding portion thereof are selected from source
In IgG1, IgG2, IgG3 and IgG4 of people.
In one embodiment of the invention, the heavy chain constant region of the antibody or its antigen-binding portion thereof is next
Derived from the IgG1 of people, the amino acid sequence of the IgG1 heavy chain constant region is for example as shown in SEQ ID NO:19.
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD
KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:19)
Encode the nucleic acid sequence of IgG1 heavy chain constant region
In one embodiment of the invention, the antibody or the constant region of light chain of its antigen-binding portion thereof derive from
People.
In one embodiment of the invention, the antibody or the constant region of light chain of its antigen-binding portion thereof be from
κ the or λ type of people.
In one embodiment of the invention, the constant region of light chain of the antibody or its antigen-binding portion thereof is next
Derived from the κ type of people, the amino acid sequence of the κ type constant region of light chain is for example as shown in SEQ ID NO:21.
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL
SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:21)
Encode the nucleic acid sequence of κ type constant region of light chain:
In one embodiment of the invention, the antibody be whole antibody, bispecific antibody, scFv, Fab, Fab',
F(ab')2Or Fv.
In one embodiment of the invention, the antibody is human antibody.
In one embodiment of the invention, the PCSK9 is people PCSK9.
In one embodiment of the invention, the anti-PCSK9 antibody or its antigen-binding portion thereof, wherein described
Anti- PCSK9 antibody be less than about 10-5M is, for example, less than about 10-6M、10-7M、10-8M、10-9M or 10-10M or smaller KD
In conjunction with PCSK9 albumen.
The invention further relates to nucleic acid molecules, contain any one of coding first aspect present invention anti-PCSK9 antibody or its
The sequence of antigen-binding portion thereof or its segment；Preferably, the nucleic acid molecules have nucleotides sequence shown in SEQ ID NO:17
Column and/or SEQ ID NO:18 shown in nucleotide sequence.
In one embodiment of the invention, the nucleic acid molecules contain the anti-of any one of coding first aspect present invention
CDR region (such as CDR1, CDR2, CDR3) sequence of the heavy chain variable region of PCSK9 antibody or its antigen-binding portion thereof.
In one embodiment of the invention, the nucleic acid molecules contain the anti-of any one of coding first aspect present invention
CDR region (such as CDR1, CDR2, CDR3) sequence of the light chain variable region of PCSK9 antibody or its antigen-binding portion thereof.
In one embodiment of the invention, the nucleic acid molecules contain the anti-of any one of coding first aspect present invention
The sequence of the heavy chain variable region of PCSK9 antibody.In specific embodiments of the present invention, the nucleic acid molecules contain such as SEQ ID
Sequence shown in NO:17.
In one embodiment of the invention, the nucleic acid molecules contain the anti-of any one of coding first aspect present invention
The sequence of the light chain variable region of PCSK9 antibody.In specific embodiments of the present invention, the nucleic acid molecules contain such as SEQ ID
Sequence shown in NO:18.
The invention further relates to carriers, contain nucleic acid molecules any one of of the invention.
The invention further relates to host cells, contain nucleic acid molecules or carrier any one of of the invention.
Another aspect of the invention is related to a kind of conjugate comprising antibody or its antigen-binding fragment and coupling portion
Point, wherein the antibody is described in any item anti-PCSK9 antibody of the invention or its antigen-binding portion thereof, the coupling moiety
For detectable label；Preferably, the coupling moiety be radioactive isotope, fluorescent material, luminescent substance, coloring matter or
Enzyme.
Another aspect of the invention is related to a kind of kit comprising anti-PCSK9 antibody described in any one of present invention
Or its antigen-binding portion thereof, or including conjugate of the invention；
Preferably, the kit further includes secondary antibody, anti-PCSK9 antibody described in specific recognition or its antigen knot
Close part；Optionally, the secondary antibody further includes detectable label, such as radioactive isotope, fluorescent material, shiner
Matter, coloring matter or enzyme.
Another aspect of the invention be related to described in any item anti-PCSK9 antibody of the invention or its antigen-binding portion thereof or
The purposes of conjugate of the invention in reagent preparation box, the kit be used to detect PCSK9 presence in the sample or its
It is horizontal.
The invention further relates to composition (such as pharmaceutical compositions), anti-containing any one of first aspect present invention
Nucleic acid molecules, carrier or the host cell of PCSK9 antibody or its antigen-binding portion thereof, any one of the present invention, and optional
Pharmaceutically acceptable carrier or excipient.
In embodiments of the invention, also contain statins in the composition.
In embodiments of the invention, the statins for example selected from Seeley Wei Siting, Atorvastatin, pungent cut down
Statin, Pitavastatin, Rosuvastatin, Fluvastatin, Lovastatin and Pravastatin.
The invention further relates to the anti-PCSK9 antibody or its antigen-binding portion thereof, the present invention of any one of first aspect present invention
Nucleic acid molecules, carrier, host cell or the composition of any one are in preparation prevention or treatment cardiovascular disease or imbalance, thrombus
The purposes of obliteran or the drug of imbalance.
In one embodiment of the invention, wherein the cardiovascular disease or imbalance are selected from dyslipidemia, coronal
Atherosclerotic heart disease, acute myocardial infarction, asymptomatic Carotid atherosis, apoplexy and periphery artery occlusion disease
Disease.
In one embodiment of the invention, wherein cholesterol of the dyslipidemia in blood increases, is sweet
Oily three rouge increase, low-density lipoprotein (LDL) increases and high-density lipoprotein (HDL) is reduced.It is specific real at of the invention one
It applies in scheme, the dyslipidemia refers to hypercholesterolemia.
In one embodiment of the invention, wherein the thrombosis or imbalance is selected from pulmonary embolism and view
Nethike embrane central vein embolism.
The invention further relates to the anti-PCSK9 antibody or its antigen-binding portion thereof, the present invention of any one of first aspect present invention
Nucleic acid molecules, carrier, host cell or the composition of any one reduce the use of the drug of lipoprotein levels in blood in preparation
On the way.
In embodiments of the invention, the lipoprotein is selected from cholesterol, triglyceride and low-density lipoprotein.
Another aspect of the invention is related to described in any item anti-PCSK9 antibody of the invention or its antigen-binding portion thereof, sheet
The nucleic acid molecules of invention, carrier of the invention, host cell of the invention, conjugate of the invention or composition of the invention
Preparing the purposes in following drug:
Drug of the specificity in conjunction with PCSK9,
Drug of the PCSK9 in conjunction with LDL-R is blocked,
The drug of LDL-R level in cell surface LDL-R quantity or blood plasma is improved,
LDL or the drug of LDL-c level in blood plasma are reduced,
Inhibit the drug of LDL accumulation in blood plasma,
The drug for the LDL-R degradation for inhibiting PCSK9 to be mediated, or
Improve the drug of cholesterol entrained by LDL and/or the metaboilic level of TG.
Another aspect of the invention is related to a kind of method in vivo or in vitro, including uses a effective amount of present invention any
Anti- PCSK9 antibody or its antigen-binding portion thereof, nucleic acid molecules of the invention, carrier of the invention, place of the invention described in
The step of chief cell, conjugate of the invention or composition of the invention, the method are selected from as follows:
Method of the specificity in conjunction with PCSK9,
PCSK9 method in conjunction with LDL-R is blocked,
The method for improving LDL-R level in cell surface LDL-R quantity or blood plasma,
The method of LDL or LDL-c level in blood plasma is reduced,
Inhibit the method for LDL accumulation in blood plasma,
The method for the LDL-R degradation for inhibiting PCSK9 to be mediated, or
The method for improving cholesterol entrained by LDL and/or the metaboilic level of TG.
The invention further relates to the method prevented or treat cardiovascular disease or imbalance, thrombosis or imbalance, institutes
The method of stating includes to the anti-PCSK9 antibody of any one of the first aspect present invention of subject in need prevention or therapeutically effective amount
Or the step of its antigen-binding portion thereof, the nucleic acid molecules of any one of the present invention, carrier, host cell or composition.
In one embodiment of the invention, wherein the cardiovascular disease or imbalance are selected from dyslipidemia, coronal
Atherosclerotic heart disease, acute myocardial infarction, asymptomatic Carotid atherosis, apoplexy and periphery artery occlusion disease
Disease.
In one embodiment of the invention, wherein cholesterol of the dyslipidemia in blood increases, is sweet
Oily three rouge increase, low-density lipoprotein (LDL) increases and high-density lipoprotein (HDL) is reduced.It is specific real at of the invention one
It applies in scheme, the dyslipidemia refers to hypercholesterolemia.
In one embodiment of the invention, wherein the thrombosis or imbalance is selected from pulmonary embolism and view
Nethike embrane central vein embolism.
The invention further relates to the methods for reducing lipoprotein levels in blood, and the method includes preventing to subject in need
Or the anti-PCSK9 antibody or its antigen-binding portion thereof, any one of the present invention of any one of first aspect present invention of therapeutically effective amount
Nucleic acid molecules, carrier, host cell or the step of composition.
In embodiments of the invention, the lipoprotein is selected from cholesterol, triglyceride and low-density lipoprotein.
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 anti-PCSK9 antibody of the invention compared with existing antibody, has higher affinity
With can more effectively reduce LDL-c in hyperlipidemia rhesus monkeys, have a good application prospect.
The present invention is described further below:
In the present invention, unless otherwise stated, Science and Technology noun used herein has art technology
The normally understood meaning of personnel institute.Also, protein used herein and nucleic acid chemistry, molecular biology, cell and tissue
Culture, microbiology, immunology relational language and laboratory operation step be in corresponding field widely used term and often
Advise step.Meanwhile for a better understanding of the present invention, the definition and explanation of relational language is provided below.
In the present invention, when referring to PCSK9 albumen (Proprotein convertase subtilisin/kexin
Type 9) amino acid sequence when comprising overall length (such as the source of people NP_777596.21, source of mouse NP_ of PCSK9 albumen
705793.1 or monkey source NP_001106130.1)；It further include its fusion protein, such as the Fc protein fragments with mouse or human IgG
The segment that (mFc or hFc) is merged.However, it will be appreciated by those skilled in the art that in the amino acid sequence of PCSK9 albumen, it can
It is naturally-produced or be artificially introduced mutation or variation (including but not limited to replace, be deleted and/or added), without influencing its biology
Function.
In the present invention, term EC50Refer to half-maximal effect concentration (concentration for 50%of maximal
Effect), refer to the concentration that can cause 50% ceiling effect.
In the present invention, term " antibody " refers to usually (each pair of to have " light " (L) chain by two pairs of identical polypeptide chains
With " weight " (H) chain) composition immunoglobulin molecules.Antibody light chain can be classified as κ and lambda light chain.Heavy chain can be classified as μ,
δ, γ, α or ε, and the isotype of antibody is defined as IgM, IgD, IgG, IgA and IgE respectively.It, can in light chain and heavy chain
Become area to connect with constant region by area " J " of about 12 or more amino acid, heavy chain also includes about 3 or more amino
Area " D " of acid.Each heavy chain is by heavy chain variable region (VH) and heavy chain constant region (CH) composition.Heavy chain constant region is by 3 structural domains
(CH1、CH2 and CH3) it forms.Each light chain is by light chain variable region (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 the various of immune system
The combination of the first component (C1q) of cell (for example, effector cell) and classical complement system.VHAnd VLArea can also be subdivided into tool
There is denatured region (referred to as complementary determining region (CDR)), is interspersed with the more conservative region for being known as framework region (FR).Respectively
VHAnd VLBy in the following order: arranged from amino terminal to carboxyl terminal 3 of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4
CDR and 4 FR composition.Variable region (the V of each heavy chain/light chain pairHAnd VL) it is respectively formed paratope.Amino acid is to each area
The distribution of domain or structural domain follows Kabat Sequences of Proteins of Immunological Interest
(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 any specific method for generating antibody.Such as comprising, particularly, recombinant antibodies, monoclonal antibody
And polyclonal antibody.Antibody can be the antibody of different isotypes, for example, IgG is (for example, IgG1, IgG2, IgG3 or IgG4 are sub-
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
The ability for the same antigen (for example, PCSK9) that part keeps binding antibody to be combined is competed with complete antibody to the special of antigen
Property combine.Usually referring to, Fundamental Immunology, Ch.7 (Paul, W., ed., second edition, Raven Press,
N.Y. (1989), are incorporation by reference in its entirety, for all purposes.By recombinant DNA technology or it can pass through
The enzymatic or chemical disruption of complete antibody generate antigen-binding portion thereof.In some cases, antigen-binding portion thereof include Fab,
Fab'、F(ab')2、It is Fd, Fv, dAb and complementary determining region (CDR) segment, single-chain antibody (for example, scFv), chimeric antibody, dual anti-
Body (diabody) and such polypeptide, it includes at least one for being enough to assign the antibody of polypeptid specificity antigen binding capacity
Point.
In the present invention, term " Fd segment " means by VHAnd CHThe antibody fragment of 1 structural domain composition；Term " Fv segment "
Mean by the V of the single armed of antibodyLAnd VHThe antibody fragment of structural domain composition；Term " dAb segment " means by VHStructural domain composition
Antibody fragment (Ward et al., Nature 341:544-546 (1989))；Term " Fab segment " means by VL、VH、CLAnd CH1 knot
The antibody fragment of structure domain composition；Term " F (ab')2Segment " means two Fab comprising connecting by the disulphide bridges on hinge area
The antibody fragment of segment.
In some cases, the antigen-binding portion thereof of antibody is single-chain antibody (for example, scFv), wherein VLAnd VHStructural domain
Connector by the way that single polypeptide chain can be produced as match to be 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)).Such scFv molecule can have general structure: NH2- VLConnector-VH- COOH or NH2- VHConnector-VL?
COOH.Suitable prior art connector (link peptide) is made of duplicate GGGGS amino acid sequence or its variant.For example, can make
With with amino acid sequence (GGGGS)4Connector, but can also be used its variant (Holliger et al. (1993),
Proc.Natl.Acad.Sci.USA90:6444-6448).Other connectors for use in the present invention are 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. description.In embodiments of the invention, the sequence of the link peptide is
(GGGGS)3。
In some cases, antibody is bispecific antibody, can be combined respectively with two kinds of antigens or epitope,
Light chain, heavy chain or its antigen-binding portion thereof including specifically binding the antibody of the first antigen, and specific binding second are anti-
Light chain, heavy chain or its antigen-binding portion thereof of former antibody.In embodiments of the invention, it is tied in the bispecific antibody
The light chain, heavy chain or its antigen-binding portion thereof for closing the antibody of the first antigen can be the antibody or its antigen knot of any one of the present invention
Part is closed, light chain, heavy chain or its antigen-binding portion thereof of the antibody of the second antigen of the specific binding can be other anti-
PCSK9 antibody or its antigen-binding portion thereof or antibody or its antigen-binding portion thereof for other antigens.
In some cases, antibody is double antibody, that is, bivalent antibody, wherein VHAnd VLStructural domain table in single polypeptide chain
Reach, but using too short connector so that do not allow to match between two structural domains of same chain, thus force structural domain with
The complementary domain of another chain match and generate 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))。
It can be used routine techniques well known by persons skilled in the art (for example, recombinant DNA technology or enzymatic or chemical disruption
Method) antigen-binding portion thereof (for example, above-mentioned antibody fragment) of antibody is obtained from given antibody (such as monoclonal antibody 2E12),
And by with for complete antibody in a manner of identical mode with regard to specificity screening antibody antigen-binding portion thereof.
In the present invention, the antigen-binding portion thereof includes single-chain antibody (scFv), chimeric antibody, double antibody, scFv-
Fc bivalent molecule, dAb and complementary determining region (CDR) segment, Fab segment, Fd segment, Fab' segment, Fv and F (ab')2Segment.
In the present invention, the IgG1 heavy chain constant region includes various allografts, as G1m (f), G1m (z), G1m (z,
Or G1m (z, a, x) a).In embodiments of the invention, the IgG1 heavy chain constant region is G1m (f) type.
In the present invention, the κ constant region of light chain includes various allografts, such as Km1, Km1,2 or Km3.In the present invention
Embodiment in, the κ constant region of light chain be Km3 type.
In the present invention, the lambda light chain constant region includes various allografts, such as λ I, λ II, λ III, λ VI.In the present invention
Embodiment in, the lambda light chain constant region be II type of λ.
Antibody nucleic acids molecule of the present invention also can use traditional genetic engineering recombinant technique or chemical synthesis side
Method obtains.On the one hand, the sequence of antibody nucleic acids molecule of the present invention contains the heavy chain variable region or anti-of anti-PCSK9 antibody
The partial nucleic acid sequence of body molecule.On the other hand, the sequence of antibody nucleic acids molecule of the present invention also includes anti-PCSK9 antibody
Light chain variable region or antibody molecule partial nucleic acid sequence.On the other hand, the sequence of antibody nucleic acids molecule of the present invention
It further include the CDR sequence of heavy chain or light chain variable region.Complementary determining region (complementary determinant region,
CDR be) position in conjunction with epitope, the CDR sequence in the present invention by IMGT/V-QUEST (http: //
Imgt.cines.fr/textes/vquest/ it) is determined.But it is different CDR sequence that division methods obtain slightly not
Together.
The present invention relates to the recombinant expression carrier for containing the nucleic acid molecules, the host for being also related to having converted these molecules is thin
Born of the same parents.Moreover, the invention further relates to cultivate and separate under given conditions using the host cell for containing the nucleic acid molecules
To the method for inventing the antibody.
Antibody amino acids sequence
Antibody 8D8F heavy chain variable amino acid sequence is SEQ ID NO:15 respectively.Antibody 8D8F light chain variable region amino
Acid sequence is SEQ ID NO:16.
The amino acid sequence determination of the CDR of the heavy chain and light chain variable region of antibody 8D8F is as follows:
The amino acid sequence of CDR1, CDR2 and CDR3 of antibody 8D8F heavy chain are respectively SEQ ID NO:1-3.
The amino acid sequence of CDR1, CDR2 and CDR3 of antibody 8D8F light chain are respectively SEQ ID NO:4-6.
The amino acid sequence determination of the FR of the heavy chain and light chain variable region of antibody 8D8F is as follows:
The sequence of antibody 8D8F heavy chain variable region FR1, FR2, FR3, FR4 are respectively SEQ ID NO:7-10.Light chain variable
The sequence of area FR1, FR2, FR3, FR4 are respectively SEQ ID NO:11-14.
On the other hand, the heavy chain of anti-PCSK9 antibody or the amino acid sequence of light chain variable region FR may be in SEQ ID
Occur the mutation of one or more amino acid on NO:7-10,11-14, increase or lack.Preferably, it is mutated, adds or lacks
The amino acid no more than 3 amino acid of mistake.It is further preferred that the amino acid of mutation, addition or missing is no more than 2 amino acid.
Most preferably, the amino acid for being mutated, adding or lacking is no more than 1 amino acid.
The amino acid of above-mentioned antibody or CDR region or framework region mutates, the variant after addition or missing is still protected
Stay the ability of specific binding people PCSK9.The present invention also includes the variant of such antigen-binding portion thereof.
Monoclonal antibody variant of the invention can be obtained by traditional gene engineering method.Those skilled in the art is complete
Know the method using nucleic acid mutation transformation DNA molecular.In addition, the nucleic acid molecules of encoding heavy chain and light chain variant can also lead to
Cross chemical synthesis acquisition.
In the present invention, for determine the algorithm of sequence identity and sequence similarity percentage be such as BLAST and
2.0 algorithm of BLAST, they describe respectively in (1977) Nucl.Acid.Res.25:3389-3402 such as Altschul and
Altschul etc. (1990) J.Mol.Biol.215:403-410.Using for example described in document or default parameters, BLAST
Amino acid sequence identity percentage of the invention is determined for BLAST 2.0.The software for executing BLAST analysis can be with
It is for the public to obtain by National Biotechnology Information Center.
In the present invention, the amino acid sequence with amino acid sequence at least 70% sequence identity include with
The substantially same polypeptide sequence of the amino acid sequence, such as when use methods described herein are (for example, by using standard parameter
BLAST analysis) when, with polypeptide sequence of the present invention compared with contain at least 70% sequence identity, preferably at least 75%, 80%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
Those of higher sequence identity sequence.
In the present invention, term " carrier ", which refers to, can be inserted the polynucleotide for encoding certain albumen and make egg
A kind of white nucleic acid delivery vehicle for obtaining expression.Carrier can make the heredity of its carrying by conversion, transduction or transfection host cell
Substance element is expressed in host cell inner expression.For example, carrier includes: plasmid；Phasmid；Coemid；Manually
The artificial chromosome (PAC) of chromosome such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or the source P1；Phagocytosis
Body such as λ bacteriophage or M13 bacteriophage and animal virus etc..Animal virus type as carrier have retrovirus (including
Slow virus), adenovirus, adeno-associated virus, herpesviral (such as herpes simplex virus), poxvirus, baculoviral, papillomatosis
Poison, papova viruses (such as SV40).A kind of element that carrier may be expressed containing various control, including promoter sequence
Column, transcriptional initiation sequence, enhancer sequence, selection element and reporter gene.In addition, carrier can also contain replication origin.
Carrier is it is also possible to include the ingredient for assisting it to enter cell, such as virion, liposome or protein coat, but not only only
There are these substances.
In the present invention, term " host cell " refers to importing the cell of carrier, including following many cell types, such as
The prokaryotic cells such as Escherichia coli or withered grass bacterium, such as yeast cells or Aspergillus fungal cell, such as S2 drosophila cell or Sf9 elder brother
Worm cell, or such as fibroblast, Chinese hamster ovary celI, COS cell, NSO cell, HeLa cell, bhk cell, 293 cell of HEK
Or the zooblast of people's cell.
Antibody fragment of the invention can use the complete antibody molecule of hydrolysis obtain (referring to Morimoto et al.,
J.Biochem.Biophys.Methods 24:107-117 (1992) and Brennan et al., Science 229:81
(1985)).In addition, these antibody fragments can also directly by recombinant host cell generate (reviewed in Hudson,
Curr.Opin.Immunol.11:548-557 (1999)；Little et al., Immunol.Today, 21:364-370
(2000)).For example, Fab' segment can directly be obtained from E.coli cell or chemical lotus root joins to form 2 segment of F (ab')
(Carter et al., Bio/Technology, 10:163-167 (1992)).For another example, F (ab')2Segment can use leucine
Zipper GCN4 connection obtains.In addition, Fv, Fab or F (ab')2Segment can also be directly from recombinant host cell culture solution directly
It is isolated.Those skilled in the art have full knowledge that the other technologies for 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 the antibody.It herein, is detection in conjunction with binding affinity of the antibody of the first antigen to second of antigen
Less than or it is very weak.In some embodiments, certain antigen-specific antibodies refer to affinity (KD)≤10- 5M (such as 10? 6M、10- 7M、10- 8M、10- 9M、10- 10M etc.) combine the antigen, wherein KDRefer to the ratio (koff/ of dissociation yield and Percentage bound
Kon), can be measured using method familiar to those skilled in the art.
In the present invention, term " KD" refer to specific antibodies-antigen interactions Dissociation equilibrium constant, it is used to describe
Binding affinity between antibody and antigen.Equilibrium dissociation constant is smaller, and antibody-antigen binding is closer, antibody and antigen it
Between affinity it is higher.In general, antibody is to be less than about 10-5M is, for example, less than about 10-6M、10-7M、10-8M、10-9M or 10-10M or smaller Dissociation equilibrium constant (KD) combine antigen.It is, for example, possible to use surface plasma body resonant vibration arts (SPR) to exist
K is measured in BIACORE instrumentD。
In embodiments of the invention, anti-PCSK9 antibody of the invention can specifically with people's PCSK9 albumen knot
It closes.
In the present invention, term " effective quantity " refers to the amount for being enough to obtain or at least partly obtaining desired effect.For example,
Prevention condition effective amount refers to, it is sufficient to prevent, prevent, or postpone the amount of the generation of disease；Treatment condition effective amount refers to, it is sufficient to
Cure or at least partly prevent suffer from disease patient disease and its complication amount.Such effective quantity is measured to exist completely
Within the limit of power of those skilled in the art.For example, therapeutical uses, which are effectively measured, will depend on disease to be treated
Severity, the overall status of the immune system of patient oneself, the ordinary circumstance of patient such as age, weight and gender, drug
Method of application, and the other treatment being administered simultaneously etc..
In embodiments of the invention, anti-PCSK9 antibody of the invention is able to suppress the combination of huPCSK9 and EGFA.
In the present invention, the blood includes whole blood, serum, blood plasma etc., the processing and preparation method of various blood samples
It is well known in the art.
In the present invention, the hypercholesterolemia includes familial hypercholesterolemia and non-familial hypercholesterolemia
Disease.
In the present invention, the dyslipidemia refers to compared with normal population, cholesterol, triglyceride in serum, low
High-density lipoprotein in one of density lipoprotein or a variety of raisings and/or serum reduces.
In the present invention, common usage is deferred in 20 kinds of conventional amino acids and its abbreviation.Referring to Immunology-A
Synthesis (second edition, E.S.Golub and D.R.Gren, Eds., Sinauer Associates, Sunderland, Mass.
(1991)) it, is incorporation by reference.
In embodiments of the invention, one plant of anti-PCSK9 monoclonal antibody is screened using phage antibody library technique.
It is compared with existing anti-PCSK9 antibody, the affinity of this strain antibody is higher, and it is intracorporal more to effectively reduce hyperlipidemia monkey
LDL-c is horizontal, it is therefore expected that they will have better curative effect, and the effective dose of drug can be reduced, to further drop
The toxic side effect of low drug.Moreover, the antibody of high-affinity has in terms of the stability of high concentration liquid preparation and druggability
Certain advantage.
Detailed description of the invention
The relative affinity of Fig. 1: monoclonal phage ELISA identification phage-Abs.
It is wherein followed successively by test antibodies, positive control (antibody mil66) and negative control from left to right in every group of column diagram
(BSA)。
The relative affinity of Fig. 2: gradient dilution phage ELISA identification phage-Abs.
Fig. 3: pTGS-1 plasmid map.
Fig. 4: (wherein DNA marker is Marker III to PCR amplification 8D-CDR1,2M-Vkappa DNA fragmentation overall length
(TIANGEN, MD103)), overall length is about 763bp.
The relative affinity of Fig. 5: gradient dilution phage ELISA identification phage-Abs.
The Binding experiment of Fig. 6: Mil66 and 8D8F and PCSK9.
Fig. 7: Mil66 and 8D8F and PCSK9 Competitive assays are tested.
The activity of cell biology that Fig. 8: Mil66 and 8D8F absorbs LDL.
Adjustment effect of Fig. 9: Mil66, the Mil68-4 antibody to LDL-c.
Adjustment effect (decline percentage) of Figure 10: Mil66, the Mil68-4 antibody to LDL-c.
Adjustment effect of Figure 11: Mil66, the Mil68-4 antibody to TC.
Adjustment effect (decline percentage) of Figure 12: Mil66, the Mil68-4 antibody to TC.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment.Those skilled in the art will manage
Solution, the following examples are merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.It is not specified in embodiment specific
Technology or conditions person, described technology or conditions are (yellow such as with reference to the work such as J. Pehanorm Brooker according to the literature in the art
" 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 production firm person is not specified in instrument, for the conventional products obtained can be bought by market.
Embodiment 1: the biopanning of anti-huPCSK9 single-chain antibody
It is the immune pipe of antigen coat with huPCSK9-his (NP_777596.2), antigen coat amount is 5 μ g/ pipe, 4 DEG C of coatings
Overnight.Immune pipe and naive phage antibody library (laboratory oneself preparation) are closed off using 4% skimmed milk power/PBST,
37 DEG C of closing 1h.Phage antibody library after closing is added in immune pipe and carries out antibody antigen combination, and the input amount of bacteriophage is about
It is 1012, 37 DEG C of reaction 1h.PBS (T) washes away unbonded bacteriophage, the HCL-Glycine antibody elution of 0.1M, with 1.5M's
Tris-HCL (pH8.8) neutralizes the bacteriophage eluted.
Phage-infect about 10mL after taking 550 μ l to neutralize grows to the TG1 bacterium solution of logarithmic growth phase, 37 DEG C of standings
30min.The 2YT culture medium of 9mL benzyl containing ammonia is added.The appropriate measurement of bacterium solution bed board cfu, 37 DEG C of culture 3h of remaining bacterium are taken out, are expanded
(pfu=1 × 10 2mL helper virus VCSM13 are added to 50mL in large volume13), 30 DEG C of shaken cultivations are stayed overnight.In next day recycling
Clearly, conventional PEG precipitating, 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 good monoclonal colonies of separation of picking output after three-wheel screens are inoculated with 1mL2YTAG
In (Ampicilline:100 μ g/ml, Glucose:2%) culture medium, 37 DEG C, 220rpm overnight incubation.It is forwarded within second day new
96 hole deep-well plates, to its logarithmic growth phase, every hole is added about 10 for culture10Helper phage VCSM13.37 DEG C of static infection
After 30min, 4000rpm, 4 DEG C of centrifugation 15min are discarded supernatant, thallus with 2YTAK (Ampicilline:100 μ g/ml,
Kanamycin:70 μ g/ml) it is resuspended and precipitates, 28 DEG C, 220rpm overnight incubation.Bacteriophage supernatant after drawing amplification carries out
ELISA identifies (referring to Fig. 1).The positive colony of acquisition is sequenced, obtains 4 kinds of different antibody sequences altogether.
Embodiment 3:phage
ELISA measures the affinity of anti-huPCSK9 single-chain antibody
The displaying and purifying that the clone obtained in embodiment 2 is carried out to monoclonal phage, carry out phage gradient dilution
The affinity of ELISA experimental identification phage-abs.
It is coated with huPCSK9 with the phosphate buffer of pH7.4,4 DEG C of coatings are overnight.PBST is washed three times, 4%milk-
37 DEG C of closing 1h of PBST.Monoclonal phage after purification is diluted in proportion in 4% skimmed milk power, 100 μ are added in every hole
Sample after l dilution, RT stand 1h.Elisa plate is washed with PBST, by the HRP-anti-M13 after the dilution of 4% skimmed milk power
Monoclonal antibody is added in elisa plate, is placed at room temperature for 1h.The colour developing of TMB colour reagent box, color development at room temperature 5min.Use 10%H2SO4
Color development stopping, 50 holes μ l/.Microplate reader 450nm Single wavelength measures OD value.Several plants of different phagocytosis filtered out as the result is shown
Body antibody can be combined with huPCSK9, and the affinity of 8D is apparently higher than other clones' (referring to fig. 2).
Embodiment 4: external affinity maturation is carried out to the anti-huPCSK9 single-chain antibody 8D filtered out
PComb3 carrier (is protected purchased from Chinese plasmid vector strain cell pnca gene using the method for series of genes clone
Hiding center) it is transformed, it is allowed to the building and expression suitable for phage antibody library: the side cloned using series of genes
Method removes the region light chain cloning region in pComb3, while to heavy chain cloning
Region is transformed, and removes the part CH1 in heavy chain cloning region, then by PCR method at more grams
NcoI and NotI restriction enzyme site is added in Long Weidianchu, makes it possible to clone and express ScFv single-chain antibody.Improved carrier life
Entitled pTGS-1, plasmid map is as shown in figure 3, and construct rite-directed mutagenesis antibody to the part VH of 8D based on pTGS-1
Library carries out antibody in vitro affinity maturation.
The building of 1.8D-CDR1,2M antibody library
Respectively using 8D as template, amplification heavy chain 8D-CDR1,2 area's genes and 8D- are reacted using rite-directed mutagenesis PCR
The area CDR3-Vkappa gene (the wherein variable region that Vkappa refers to light chain).QIAgene plastic recovery kit recycles PCR piece
Section.The DNA fragmentation that above-mentioned two parts PCR reaction obtains is subjected to Overlap PCR, expands 8D-CDR1,2M-Vkappa is complete
It is 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 plastic recovery kit recycles PCR fragment (referring to fig. 4).
Plasmid pTGS-1 and 8D-CDR1,2M-Vkappa PCR overall length is carried out respectively with NcoI-HF and NotI double
Digestion.Plasmid pTGS-1 is through 1.5% agarose gel electrophoresis, gel extraction.PCR fragment uses QIAgene PCR
Purification kit kit is recycled.8:1 ratio passes through in molar ratio for PCR fragment after the recovery and pTGS-1 plasmid
The connection of T4DNA ligase room temperature is overnight.Junction fragment electric shocking method is converted into TG1 competence.It is trained using 37 DEG C of SOC culture medium
Support 1h recovery bacterium.Certain bacterium solution is taken, plate is applied, calculates storage capacity.Remaining bacterium solution 4000rpm, is centrifuged 15min at room temperature.It abandons
Supernatant, precipitating are coated on 2~3 2YTCG massive plates, 37 DEG C of inversion overnight incubations.
Antibody library storage capacity is about 104, 10 clones of random picking carry out sequence analysis from the antibody library, and accuracy is
70%.
2. the screening of the biopanning and positive colony of phage antibody library
By the antibody library of above-mentioned building, phage displaying, purifying and precipitating are carried out.Then the anti-huPCSK9 of elutriation from the library
Single-chain antibody.Amp resistance with embodiment 1, is only substituted for Cm resistance by the biopanning method of phage antibody library, and (chlorine is mould
Element: Chloramphenicol), helper virus replaces with M13KO7 by VCSM13.Anti- huPCSK9 single-chain antibody positive colony
Amp resistance is only substituted for Cm resistance with embodiment 2 by screening technique, and helper virus replaces with M13KO7 by VCSM13.Screening
To 1 plant of positive monoclonal, the anti-huPCSK9 antibody of expressed sequence unlike the prior art is named as 8D8F.
The affinity of 3.phage ELISA measurement 8D8F single-chain antibody
8D8F is carried out to the displaying and purifying of monoclonal pahge, phage gradient dilution ELISA experimental identification phage-
The affinity of abs, method is the same as embodiment 3.The results show that the affinity of 8D8F is slightly below 8D (referring to Fig. 5).
The amino acid and nucleotide sequence of 8D8F antibody are as follows:
8D8F VH
EVQLVESGGGLVQPGGSLRLSCAASGFAFGGYAMNWVRQAPGKGLDWVSTISGSGGSTNYADSVKGRF
IISRDSSKHTLYLQMNSLRAEDTAVYYCAKDSNWGNFDLWGRGTLVTVSS(SEQ ID NO:15)
Encode the nucleic acid sequence of 8D8F VH
GAGGTGCAGCTGGTGGAGAGCGGTGGTGGCCTGGTGCAGCCGGGCGGCAGCCTGCGTCTGAGCTGCGC
CGCCAGCGGCTTCGCTTTCGGCGGTTACGCTATGAACTGGGTGCGCCAGGCCCCTGGCAAGGGCCTGGACTGGGTG
AGCACCATTAGCGGTAGCGGTGGCAGCACCAACTACGCCGACAGCGTGAAGGGCCGTTTCATCATCAGCCGCGACA
GCAGCAAGCACACCCTGTACCTGCAGATGAACAGCCTGCGTGCCGAGGACACCGCCGTGTACTACTGCGCCAAGGA
CAGCAACTGGGGCAACTTCGACCTGTGGGGCCGCGGCACCCTGGTGACCGTGAGCAGC(SEQ ID NO:17)
8D8F VL
DIVMTQSPDSLAVSLGERATINCKSSESVMYRRNARNFLGWYQQKPGQPPNLLIYWASTRESGVPDRF
SGSGSGTDFTLTISSLQAEDVAVYYCQQYYTHPYTFGQGTKLEIK(SEQ ID NO:16)
Encode the nucleic acid sequence of 8D8F VL
GACATCGTGATGACCCAGAGCCCGGACAGCCTGGCCGTGAGCCTGGGCGAGCGCGCCACCATCAACTG
CAAGAGCAGCGAAAGCGTGATGTACCGTCGCAACGCTCGCAACTTCCTGGGCTGGTACCAGCAGAAGCCTGGCCAG
CCTCCTAACCTGCTGATCTACTGGGCCAGCACCCGTGAGAGCGGCGTGCCTGACCGCTTCTCTGGTAGCGGCTCTG
GCACCGACTTCACCCTGACCATCAGCAGCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCAGCAGTACTACAC
CCACCCTTACACCTTCGGCCAGGGCACCAAGCTGGAGATCAAG(SEQ ID NO:18)
Part in the above sequence with 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
The light-chain variable region gene of 8D8F and heavy chain variable region gene are cloned into equipped with constant region of light chain and light chain constant
(heavy chain constant region is the constant region of human IgG1, and amino acid sequence is as shown in SEQ ID NO:19, nucleotide sequence such as SEQ in area
Shown in ID NO:20；The constant region of constant region of light chain behaviour kappa, amino acid sequence is as shown in SEQ ID NO:21, nucleosides
Acid sequence is as shown in SEQ ID NO:22) on the pCDNA3.1 carrier of gene, transfection CHO-K1 cell (it is purchased from U.S. ATCC,
CCL-61TM), whole antibody secreting, expressing is carried out, is purified by protein A, obtains whole antibody albumen after super filter tube ultrafiltration.With
Mil66 is that (identical with Sino phenanthrene/Alirocumab sequence of regeneration member, the expression according to 8D8F antibody is pure for positive control
Change method obtains its expression and purification；Its sequence is also referred to INN 9620_H, INN 9620_L.), to being obtained 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
It is coated with PCSK9 antigen with PBS, peridium concentration is 5 μ g/ml, and 4 DEG C of coatings are overnight.PBST washes elisa plate, altogether three times.
37 DEG C of closing 1h of 1%BSA-PBST.The molar ratio of 8D8F and Mil66 and PCSK9 is since 1:1, three times gradient dilution, when dense
When degree is to 41.2ng/ μ l, two-fold dilution is carried out.Each sample does 11 gradient dilutions, last hole is as negative control.It will be dilute
The sample released is added in elisa plate, 100 holes μ l/, 37 DEG C of incubation 1h.It is washed three times with 200 μ l/ hole PBST, by HRP label
Goat anti-Human IgG secondary antibody is diluted in 1%BSA-PBST by 1:2000,37 DEG C of incubation 1h.The colour developing of TMB colour reagent box, 100 μ
The hole l/, color development at room temperature 5min.Use 10%H2SO4Color development stopping, 50 holes μ l/.It is read under the wavelength of 450nm.
As a result such as Fig. 6 and following table 1.
EC of table 1:8D8F and the Mil66 antibody in conjunction with PCSK950Value
| Mil66 | 8D8F | |
| EC50(ng/ml) | 45.72 | 6.58 |
The results show that 8D8F can be in conjunction with PCSK9 with Mil66.But the binding ability of 8D8F is apparently higher than Mil66,
Its medium effective concentration EC50Value is than 6.6 times of Mil66 high or so.
The affinity of 2.BIAcore measurement 8D8F whole antibody
Using the affinity of prize law measurement whole antibody.By anti-human FCOn the antibody coupling to the surface of chip CM5 of section, point
Not Xi Shi Mil66,8D8F antibody to 1 μ g/ml, guarantee about 100RU antibody by the antibody capture of anti-human Fc.By huPCSK9-his
A series of concentration gradient (200nm, 100nm, 50nm, 25nm, 12.5nm, 6.25nm) is set and flows through fixed phase surface, is measured
The affinity of antibody.As a result as shown in Table 2 below.
Table 2:Mil66 and 8D8F affinity of antibody constant measuring
The result shows that the K of 8D8F antibodyDValue improves about 7.7 times than Mil66.
3.8D8F whole antibody and PCSK9 Competitive assays are tested
It is coated with EGFA-Fc with the carbonate buffer solution of pH9.6, peridium concentration is 2 μ g/ml, and 4 DEG C of coatings are overnight.PBST is washed
It washs three times, 37 DEG C of closing 1h of 1%BSA-PBST.It is dilute that then 3 times of gradients are carried out with 1%BSA-PBST dilution antibody to 10 μ g/ml
It releases, 10 dilution gradients is set altogether, then to the huPCSK9-his that 8 isometric μ g/ml are added in the sample diluted, 37
DEG C be incubated for 1h.Then the mixture being incubated for is added in elisa plate, 37 DEG C of incubation 1h.It is washed three times with 200 μ l/ hole PBST, it will
The anti-His secondary antibody of mouse of HRP label is diluted in 1%BSA-PBST by 1:3000,37 DEG C of incubation 1h.TMB colour reagent box is aobvious
Color, 100 holes μ l/, color development at room temperature 8min.Use 10%H2SO4Color development stopping, 50 holes μ l/.450nm reading.As a result such as Fig. 7 and following
Table 3.
Table 3Mil66,8D8F and PCSK9 Competitive assays IC50Value
| Mil66 | 8D8F | |
| IC50(ng/ml) | 15.43 | 30.9 |
The results show that Mil66 and 8D8F can inhibit the combination of huPCSK9 and LDLR.
The activity of cell biology that 4.Mil66,8D8F absorb LDL
HepG2 cell is inoculated on 96 orifice plates with the amount in 40000/hole with complete medium (DMEM), culture plate is put
To 37 DEG C of cell incubator (5%CO2) in culture for 24 hours after, be sucked out supernatant, be added the DMEM culture medium containing 1%FBS continue to cultivate
16h.After anti-PCSK9 antibody (Mil66,8D8F) is diluted to 400 μ g/ml, 2 times of gradient dilutions dilute 8 concentration altogether,
HuPCSK9-his is diluted to 800 μ g/ml simultaneously, the antibody and antigen diluted is mixed with the ratio of 1:1,37 DEG C of cell trainings
It supports case to be incubated for 30 minutes, supernatant in 96 orifice plates is then sucked out, the antigen-antibody mixture after being incubated for is added, while LDL is added,
Tissue culture plate is put into 37 DEG C of cell incubators and supernatant is sucked out after cultivating 6 hours by the final concentration of 10 μ g/ml of LDL, PBS
After twice of cleaning, culture plate is put into fluorescence intensity in microwell plate detection system, and a length of 485nm of excitation light wave emits light wave
A length of 535nm.Using antibody concentration as abscissa, Reversal of PCSK9effect on LDL uptake (%) is vertical sits
Mark, fitting quadruplex parameters curve calculate EC50Value.Mil66 is added as control in each plate when sample detection.
As a result as shown in Figure 8.
The results show that Mil66,8D8F can effectively prevent the combination of huPCSK9-his and LDLR, to reverse
The inhibiting effect that huPCSK9-his absorbs LDL.
The regulating plasma lipid pharmacodynamic test of embodiment 6:Mil66,8D8F to hyperlipidemia rhesus macaque
8D8F is renamed as Mil68-4.Mil66 and Mil68-4 antibody is evaluated to the blood of hyperlipidemia rhesus macaque
Rouge adjustment effect.
This, which is tested, is selected in 15 hyperlipidemia rhesus macaquies, weight 7-15kg, the internal horizontal 1.1-1.7mmol/ of LDL-c
L.Monkey is divided into 2 groups: Mil68-4 (1.2mg/kg) and MIL66 (1.2mg/kg) group, every group of 3 hyperlipidemia rhesus macaquies are single
Secondary subcutaneous administrations.It acquires respectively before administration blood sample 2 times, then respectively acquires blood sample one within 1,3,5,7,14 day upon administration
It is secondary, it is tested and analyzed for blood lipids index, it may be assumed that total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL-c), high density
Lipoprotein (HDL-c).Before sampling operation, fasting 14-16h is not anaesthetized animal, through forearm venous blood collection 0.5ml.Use one
Roche Cobas C501 detects blood lipid indices.
Experimental result is as shown in Fig. 9 to Figure 12.
The results show that (1.2mg/kg is equivalent to clinical dosage 42mg/ people), MIL66 and Mil68-4 under Isodose
Similar to the action time of LDL-c after drug single-dose, first day LDL-c is remarkably decreased upon administration, and downward trend
5-7 days after being continued for administration.Wherein Mil68-4 was in D5-D7 days reduction 51.95%-49.45%, and Mil66 exists
D5-D7 days reduction 44.05%-40.87%.The drug action of Mil66 is weakened after D7 days, is fallen within D14 days
21.73%.And the drug action of Mil68-4 can continue to 14 days, LDL-c reduces by 44.34% within D14 days.
As can be seen from the above results, Mil68-4 is substantially better than positive control to the action intensity of LDL-c and duration
Mil66 (Fig. 9,10).It is suitable to the adjustment effect of hyperlipidemia rhesus macaque TC after MIL66 and Mil68-4 single-dose.With baseline
Value compares, TC day lasting reduction from first day to the 5th.In terms of the 1st, 3,5 day data, the intensity that MiL66 reduces TC is slightly better than
Mil68-4, but Mil68-4 drug action can continue to the 14th day, and Mil66 weakens (ginseng from drug action after the 7th day
See Figure 11,12).Obvious effect there are no to HDL-c and TG after Mil66 and Mil68-4 drug single-dose.
Although a specific embodiment of the invention has obtained detailed description, it will be understood to those of skill in the art that.Root
According to all introductions having disclosed, those details can be carry out various modifications and be replaced, these change in guarantor of the invention
Within the scope of shield.Full scope of the invention is given by the appended claims and any equivalents thereof.
Claims (37)
1. anti-PCSK9 antibody or its antigen-binding portion thereof, in which:
The heavy chain variable region of the anti-PCSK9 antibody includes: the amino acid sequence V as shown in SEQ ID NOs:1-3 respectivelyH
CDR1-VHCDR3；
With
The light chain variable region of the anti-PCSK9 antibody includes: the amino acid sequence V as shown in SEQ ID NOs:4-6 respectivelyL
CDR1-VL CDR3。
2. anti-PCSK9 antibody according to claim 1 or its antigen-binding portion thereof, heavy chain variable region FR1, FR2, FR3,
The area FR4 separately includes the sequence as shown in SEQ ID NOs:7-10；
With
The area its light chain variable region FR1, FR2, FR3, FR4 separately includes the sequence as shown in SEQ ID NOs:11-14.
3. anti-PCSK9 antibody or its antigen-binding portion thereof described in any claim, heavy chain in -2 according to claim 1
Constant region is selected from IgG, IgM, IgE, IgD and IgA from people.
4. anti-PCSK9 antibody according to claim 3 or its antigen-binding portion thereof, wherein the IgG be IgG1, IgG2,
IgG3 or IgG4.
5. anti-PCSK9 antibody or its antigen-binding portion thereof described in any claim, light chain in -2 according to claim 1
Constant region is the κ or λ from people.
6. anti-PCSK9 antibody or its antigen-binding portion thereof described in any claim in -2 according to claim 1, are complete
Antibody, bispecific antibody, scFv, Fab, Fab', F (ab')2Or Fv.
7. according to claim 1 to anti-PCSK9 antibody described in any claim in 2 or its antigen-binding portion thereof, wherein institute
The anti-PCSK9 antibody stated is less than 10-5The K of MDIn conjunction with PCSK9 albumen.
8. according to claim 1 to anti-PCSK9 antibody described in any claim in 2 or its antigen-binding portion thereof, wherein institute
The anti-PCSK9 antibody stated is less than 10-6The K of MDIn conjunction with PCSK9 albumen.
9. according to claim 1 to anti-PCSK9 antibody described in any claim in 2 or its antigen-binding portion thereof, wherein institute
The anti-PCSK9 antibody stated is less than 10-7The K of MDIn conjunction with PCSK9 albumen.
10. according to claim 1 to anti-PCSK9 antibody described in any claim in 2 or its antigen-binding portion thereof, wherein
The anti-PCSK9 antibody is less than 10-8The K of MDIn conjunction with PCSK9 albumen.
11. according to claim 1 to anti-PCSK9 antibody described in any claim in 2 or its antigen-binding portion thereof, wherein
The anti-PCSK9 antibody is with 10-9The K of M number gradeDIn conjunction with PCSK9 albumen.
12. a kind of nucleic acid molecules, contain in coding claim 1-11 anti-PCSK9 antibody described in any claim or
Its antigen-binding portion thereof.
13. nucleic acid molecules according to claim 12, wherein the nucleic acid molecules have core shown in SEQ ID NO:17
Nucleotide sequence.
14. nucleic acid molecules according to claim 12, wherein the nucleic acid molecules have core shown in SEQ ID NO:18
Nucleotide sequence.
15. a kind of carrier contains nucleic acid molecules described in any claim in claim 12 to 14.
16. a kind of host cell is wanted containing nucleic acid molecules described in any claim in claim 12 to 14 or right
Carrier described in asking 15.
17. a kind of conjugate comprising antibody or its antigen-binding fragment and coupling moiety, wherein the antibody is right
It is required that anti-PCSK9 antibody or its antigen-binding portion thereof, the coupling moiety described in any claim are detectable in 1 to 11
Label.
18. conjugate according to claim 17, wherein the coupling moiety is radioactive isotope, luminescent substance, has
Color substance or enzyme.
19. conjugate according to claim 17, wherein the coupling moiety is fluorescent material.
20. a kind of kit comprising anti-PCSK9 antibody or its antigen described in any claim in claim 1 to 11
Bound fraction, or including conjugate described in any claim in claim 17 to 19.
21. kit according to claim 20, wherein the kit further includes secondary antibody, specific recognition
The anti-PCSK9 antibody or its antigen-binding portion thereof.
22. kit according to claim 20, wherein the secondary antibody further includes detectable label.
23. kit according to claim 22, wherein it is described it is detectable label be, shiner
Matter, coloring matter or enzyme.
24. kit according to claim 22, wherein it is described it is detectable label be.
25. anti-PCSK9 antibody or its antigen-binding portion thereof or right described in any claim are wanted in claim 1 to 11
Purposes of the conjugate described in any claim in reagent preparation box in 17 to 19 is sought, the kit is for detecting
The presence or its level of PCSK9 in the sample.
26. a kind of pharmaceutical composition, containing anti-PCSK9 antibody described in any claim in claim 1-11 or its
Conjugate described in any claim in antigen-binding portion thereof or claim 17 to 19.
27. pharmaceutical composition according to claim 26 also includes pharmaceutically acceptable carrier.
28. pharmaceutical composition according to claim 26 also includes pharmaceutically acceptable excipient.
29. the pharmaceutical composition according to any claim in claim 26 to 28, also contains statins.
30. pharmaceutical composition according to claim 29, wherein the statins are selected from Seeley Wei Siting, atropic
Cut down one of statin, Simvastatin, Pitavastatin, Rosuvastatin, Fluvastatin, Lovastatin and Pravastatin or more
Kind.
31. anti-PCSK9 antibody or its antigen-binding portion thereof or right described in any claim are wanted in claim 1-11
Conjugate described in any claim in 17 to 19 is asked to prepare prevention or treatment cardiovascular disease or rhomboembolia type disease
Purposes in the drug of disease.
32. purposes according to claim 31, wherein the cardiovascular disease is selected from dyslipidemia, coronary sclerosis
Property heart disease, acute myocardial infarction, asymptomatic Carotid atherosis, apoplexy and Peripheral arterial occlusive disease.
33. purposes according to claim 32, wherein cholesterol of the dyslipidemia in blood increases, glycerol
Three rouge increase, low-density lipoprotein increases and high-density lipoprotein reduces.
34. purposes according to claim 32, wherein the thrombosis is in pulmonary embolism and retina
Entreat venous embolism.
35. the anti-PCSK9 antibody of any claim or its antigen-binding portion thereof or claim 17 in claim 1-11
The purposes in blood in the drug of lipoprotein levels is reduced in preparation to conjugate described in any claim in 19.
36. the anti-PCSK9 antibody of any claim or its antigen-binding portion thereof or claim 17 in claim 1-11
The purposes in following drug is being prepared to conjugate described in any claim in 19:
Drug of the specificity in conjunction with PCSK9,
Drug of the PCSK9 in conjunction with LDL-R is blocked,
The drug of LDL-R level in cell surface LDL-R quantity or blood plasma is improved,
LDL or the drug of LDL-c level in blood plasma are reduced,
Inhibit the drug of LDL accumulation in blood plasma,
The drug for the LDL-R degradation for inhibiting PCSK9 to be mediated, or
Improve the drug of cholesterol entrained by LDL and/or the metaboilic level of TG.
37. a kind of method in vitro, the anti-PCSK9 including using any claim in a effective amount of claim 1-11
In antibody or its antigen-binding portion thereof or claim 17 to 19 the step of conjugate described in any claim, the side
Method is selected from as follows:
Method of the specificity in conjunction with PCSK9,
PCSK9 method in conjunction with LDL-R is blocked,
The method for improving LDL-R level in cell surface LDL-R quantity or blood plasma,
The method of LDL or LDL-c level in blood plasma is reduced,
Inhibit the method for LDL accumulation in blood plasma,
The method for the LDL-R degradation for inhibiting PCSK9 to be mediated, or
The method for improving cholesterol entrained by LDL and/or the metaboilic level of TG.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510415198 | 2015-07-15 | ||
| CN2015104151984 | 2015-07-15 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106084058A CN106084058A (en) | 2016-11-09 |
| CN106084058B true CN106084058B (en) | 2019-08-27 |
Family
ID=57219588
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610540921.6A Active CN106084058B (en) | 2015-07-15 | 2016-07-11 | Anti-human PCSK9 monoclonal antibody |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106084058B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108239150A (en) | 2016-12-24 | 2018-07-03 | 信达生物制药(苏州)有限公司 | Anti- PCSK9 antibody and application thereof |
| WO2018228406A1 (en) * | 2017-06-14 | 2018-12-20 | 江苏恒瑞医药股份有限公司 | Pcsk9 antibody, antigen-binding fragment and medical use thereof |
| CN109762067B (en) * | 2019-01-17 | 2020-02-28 | 北京天广实生物技术股份有限公司 | Antibodies that bind human Claudin18.2 and uses thereof |
| EP4063506A4 (en) * | 2019-09-19 | 2023-09-06 | Innovent Biologics (Suzhou) Co., Ltd. | Method for preventing or treating cholesterol-related diseases by using anti-pcsk9 antibody |
| CN114438075A (en) * | 2022-02-25 | 2022-05-06 | 刘博巽 | Screening method and application of bacillus subtilis proprotease 9 targeted binding protein |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102245641A (en) * | 2008-12-15 | 2011-11-16 | 瑞泽恩制药公司 | High affinity human antibodies to PCSK9 |
| CN102652139A (en) * | 2009-12-11 | 2012-08-29 | Irm责任有限公司 | Pcsk9 antagonists |
| CN104540852A (en) * | 2012-08-13 | 2015-04-22 | 瑞泽恩制药公司 | Anti-pcsk9 antibodies with ph-dependent binding characteristics |
-
2016
- 2016-07-11 CN CN201610540921.6A patent/CN106084058B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102245641A (en) * | 2008-12-15 | 2011-11-16 | 瑞泽恩制药公司 | High affinity human antibodies to PCSK9 |
| CN102652139A (en) * | 2009-12-11 | 2012-08-29 | Irm责任有限公司 | Pcsk9 antagonists |
| CN104540852A (en) * | 2012-08-13 | 2015-04-22 | 瑞泽恩制药公司 | Anti-pcsk9 antibodies with ph-dependent binding characteristics |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106084058A (en) | 2016-11-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106084058B (en) | Anti-human PCSK9 monoclonal antibody | |
| CN109937212B (en) | B7-H3 antibody, antigen binding fragment thereof and medical application thereof | |
| CN105820251B (en) | Bispecific antibody or antibody mixture with common light chain | |
| JP6734467B2 (en) | Anti-PCSK9 monoclonal antibody | |
| US10858446B2 (en) | Anti-PCSK9 antibodies and uses thereof | |
| US11402383B2 (en) | Nucleic acid encoding PCSK9 antibody | |
| CN105968200A (en) | Anti-human pd-l1 humanized monoclonal antibody and application thereof | |
| CN106008714A (en) | Anti-human pd-1 humanized monoclonal antibody and application thereof | |
| EP0629240A1 (en) | Altered antibodies, products and processes relating thereto | |
| CN101679506A (en) | Compositions and methods for binding sphingosine-1-phosphate | |
| CN102405236A (en) | Improved anti-tnfr1 polypeptides, antibody variable domains & antagonists | |
| JP2019089772A (en) | Modified antibodies and methods of making them | |
| US9090994B2 (en) | Antibody humanization by framework assembly | |
| US20230138315A1 (en) | Anti-angptl3 antibody and use thereof | |
| WO2017114230A1 (en) | Pcsk9 antibody, antigen-binding fragment thereof, and medicinal application thereof | |
| Park et al. | Generation and characterization of a novel tetravalent bispecific antibody that binds to hepatitis B virus surface antigens | |
| WO2009096162A1 (en) | Composition for neutralizing botulinus toxin type-a, and human anti-botulinus toxin type-a antibody | |
| CN105061596B (en) | The monoclonal antibody and its application of human B lymphocyte stimulating factor | |
| WO2021136773A1 (en) | Ada-response specification assay | |
| KR102115300B1 (en) | Antibody library and Screening Method of Antibody by Using the Same | |
| CA3152860A1 (en) | Anti-tfpi monoclonal antibodies | |
| CN106554420A (en) | PCSK9 antibody, its Fab and its medical usage | |
| CN117304313B (en) | Fully human monoclonal antibody specifically binding yersinia pestis V antigen | |
| WO2022218277A1 (en) | Antibody against carboxyl terminal of fgf21, and use thereof | |
| EP4201961A1 (en) | Antibody specifically bound to glycosylated ceacam5 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |