CN105037554B - The preparation and application thereof of anti-human PCSK9 antibody - Google Patents
The preparation and application thereof of anti-human PCSK9 antibody Download PDFInfo
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Abstract
The invention discloses a kind of anti-human 9 (PCSK9) chimeric antibody of proprotein convertases subtilisin and its preparation and uses.The present invention expands mouse light chain and heavy chain variable region gene respectively from mouse hybridoma, and chimeric with the light chain of human IgG and weight chain constant area gene respectively, obtains human mouse chimeric antibody by recombinant expression.Human mouse chimeric antibody of the present invention and people PCSK9 have good compatibility, and obvious inhibition PCSK9 is to the degrading activity of LDL Receptor Activity of Hepatocytes (LDLR), intake of the liver cell to LDL- cholesterol (LDL-C) is improved, blood cholesterol levels are reduced.
Description
Technical field
The present invention relates to genetic engineering fields, and in particular to a kind of preparation and application thereof of novel anti-human PCSK9 antibody.
Background technique
Proprotein convertases subtilisin 9 (PCSK9) is the of kexin sample proprotein convertases subtilisin family
9 members are made of 692 amino acid.PCSK9 is mainly derived from liver, kidney and small intestine.The synthesis of PCSK9 occurs in endoplasmic reticulum,
Blood is secreted into after a series of modifications by ER transport to golgiosome, and in golgiosome.It is now recognized that only liver
The PCSK9 of dirty secretion can be secreted into blood, and combine with the LDL receptor of surface of hepatocytes (LDLR), mediate LDLR
Degradation adjusts blood plasma cholesterol level.As the negative regulator agent of LDL receptor (LDL-R), excessive PCSK9 and liver are thin
Cellular surface LDL-R can accelerate its degradation after combining, and cause liver cell to decline the intake of LDL- cholesterol (LDL-C), Jin Erzeng
Add the LDL-C of circulation horizontal, finally increases Blood Cholesterol level.Therefore, PCSK9 has become treatment hypercholesteremia
The important target spot of disease.
Hypercholesterolemia is divided into familial and non-familial, but belongs to lipid-metabolism disease, is coronary artery disease
The important risk factor of generation.Statins be currently treatment hypercholesterolemia drug the most successful, but it is some cannot
Target cannot be reached always by being resistant to statins and the horizontal excessively high patient of LDL, lipid level.Therefore, it also needs
Develop novel fat-reducing medicament treatment hypercholesterolemia alone or synergistically.Anti- PCSK9 has become reduction blood
A kind of novel therapeutic means of liquid elevated cholesterol.So far, external to have developed multiple therapeutic monoclonal antibodies, and facing
Good curative effect is shown in bed test, as Sino phenanthrene and Regeneron unite the alirocumab developed, the similar production of Amgen
Product evolocumab, significant decrease health volunteer, familial hyperlipidemia patient and non-familial hypercholesterolemia suffer from
The LDL-C of person is horizontal；The patient ineffective for statins, its blood LDL-C is horizontal after injecting monoclonal antibody
Also 40%-72% is reduced.It is expected that PCSK9 mab treatment drug will successively obtain U.S. FDA approval in 2015-2017
Listing, this will open up the new milestone for the treatment of hypercholesterolemia.
The present invention provides the chimeric antibody of anti-PCSK9 a kind of, which can specifically combine people PCSK9, from
And effectively inhibit PCSK9 active, it is final to play the effect for reducing plasma cholesterol.
Summary of the invention
An object of the present invention is to provide the recombined chimeric antibody and its and its encoding gene of a kind of anti-PCSK9.It has
Body sequence is as follows:
Chain variable region amino acid is as described in the SEQ ID NO:1 in sequence table；
Heavy chain variable amino acid is as described in the SEQ ID NO:3 in sequence table；
Constant region of light chain amino acid is as described in the SEQ ID NO:5 in sequence table；
The amino acid of IgG1 heavy chain constant region is as described in the SEQ ID NO:9 in sequence table；
The amino acid of IgG4 heavy chain constant region is as described in the SEQ ID NO:7 in sequence table；
It is as follows to encode the above-mentioned fusion protein nucleotide sequence:
Light chain variable region nucleotide is as described in the SEQ ID NO:2 in sequence table；
Heavy chain variable region nucleotide is as described in the SEQ ID NO:4 in sequence table；
Constant region of light chain nucleotide is as described in the SEQ ID NO:6 in sequence table；
IgG1 heavy chain constant region nucleotide is as described in the SEQ ID NO:10 in sequence table；
IgG4 heavy chain constant region nucleotide is as described in the SEQ ID NO:8 in sequence table；
The third object of the present invention is to provide the preparation method of chimeric antibody
1) the human cloning PCSK9 antibody gene from positive hybridoma cell；
2) chimeric antibody construction of eukaryotic expression vector；
3) select CHO-K1, CHO-DG44 or CHO-S cell as host cell expression chimeric antibody.
Invention also provides the expression vector of above-mentioned nucleic acid sequence, expression vector includes a variety of commercial carriers or warp
The commercialization carrier for expression of eukaryon of transformation is crossed, includes pCDNA4.1, pCHO1.0, UCOE Expression Vector-Mouse
3.2kb etc., it is preferably engineered after UCOE Expression Vector-Mouse 3.2kb, the above-mentioned expression vector is introduced into conjunctions
Suitable expression system, the expression system are eukaryotic expression system, they are including but not limited to mammalian cell, yeast, elder brother
Worm cell etc..It can be used for there are many mammalian cells that protein is expressed on a large scale, such as 293 cells, Chinese hamster ovary celI, SP20 are thin
Born of the same parents, NS0 cell, COS cell, bhk cell or PerC6 cell etc., preferably Chinese hamster ovary celI；There are many ways to transfecting cell, wherein
Including but not limited to: electroporation, liposome mediated-method, calcium mediated method etc., preferably electroporation.
The expression way of the chimeric antibody is to carry out gene to recombinant vector in the host cell of stable transfection
Amplification, to improve the expression quantity of corresponding recombinant protein, for example (,) it is stable with the recombinant vector containing dihyrofolate reductase (DHFR)
After transfection lacks the host cell of DHFR, the concentration of methotrexate (MTX) can be added in cell culture fluid to expand recombination and carry
Number of copies of the body in host cell.Containing the chimeric antibody expression after, can with enzyme linked immunosorbent assay (ELISA) or its
Protein concentration in his method measurement culture solution.Chimeric antibody albumen can be purified with Mabselect SURE affinity chromatography.
The third object of the present invention is to provide the purposes of chimeric antibody
Chimeric antibody provided by the invention is used for the prevention and treatment by the PCSK9 metabolic disease induced or symptom.Its
The disease or symptom that middle PCSK9 induces are hypercholesterolemia, hyperlipemia, need the high gallbladder of LDL plasma exchange, heterozygous familial
Sterol mass formed by blood stasis, inhibin intolerance, inhibin be unable to control, the danger with hypercholesterolemia is suffered from, dyslipidemia, bile
Smoulder type hepatopathy, nephrotic syndrome, hypothyroidism, obesity, atherosclerosis and cardiovascular disease.
Detailed description of the invention
Figure 1B P4030 light chain expression vector map
Fig. 2 BP4031 heavy chain expression vector map
Fig. 3 BP4032 heavy chain expression vector map
Influence of Fig. 4 chimeric antibody to people PCSK9 degradation liver cell LDLR
Fig. 5 chimeric antibody mediates the influence of liver cell intake LDL-C to PCSK9
The linear relationship of Fig. 6 chimeric antibody quantitative detection people PCSK9
Specific embodiment
Following embodiment is provided to be further explained the present invention.It should be appreciated that these embodiments are merely to illustrate this hair
It is bright rather than have any restrictions to the present invention.Those skilled in the art are under the enlightenment of this specification to made in present invention implementation
Any variation will all be fallen within the scope of the appended claims.
The preparation of the anti-hPCSK9 chimeric antibody of embodiment 1
1, the acquisition of anti-hPCSK9 monoclonal antibody sequences
1.1 hPCSK9 are immunized mouse hybridoma cell system and obtain
Using hPCSK9 and quickantibody-mouse 3W immunologic adjuvant (KX0210041, Beijing health green spring biology section
Skill Co., Ltd) mixing in equal volume, the final concentration of 200 μ g/ml of PCSK9, immune Balb/c cell, using intramuscular injection, 100 μ
The every mouse of l/, adaptive immune splenocyte.It takes immune spleen cell to merge with Sp/20 (murine myeloma cell), is selectively trained through HAT
It supports, by semisolid culturemedium culture, filters out anti-hPCSK9 positive hybridoma cell system, screened using primary subclone,
It is final to obtain the monoclonal hybridoma system (4-2B4-4 cell line) that secrete anti-hPCSK9 antibody protein.
The acquisition of the 1.2 anti-antibody variable sequences hPCSK9
Using anti-PCSK9 antibody monoclonal cell line, cell is collected by centrifugation respectively, extracts total serum IgE, benefit with TRIZOL method
With reverse transcription reagent box (PrimeScriptTM1st strand cDNA Synthesis Kit, 6110B, Takara) it obtains
CDNA obtains antibody variable sequences by PCR amplification method, PCR reaction system (50 μ l of total volume): 10 × Buffer, 5 μ l,
Each 1 μ l (primer sequence is shown in Table 1) of 2 μ l of dNTP, upstream and downstream primer, template are 0.2 μ of (4-2B4-4 cell line) cDNA, rTaq enzyme
L is finally mended with distilled water to 50 μ l；Reaction condition: 95 DEG C of initial denaturation 3min, 95 DEG C of 15s, 52 DEG C of 30s, 72 DEG C of 30s, 33 are followed
Ring, last 72 DEG C of 15min.It obtains heavy and light chain variable region gene segment and is respectively designated as 20H (sequence in amino acid sequence such as sequence table
Shown in column 3, nucleic acid sequence is as shown in sequence 4 in sequence table) and 21K (amino acid sequence as shown in sequence 1 in sequence table, nucleic acid
Sequence is as shown in sequence 2 in sequence table).
1 antibody variable zone amplication primer of table
The building of 1.3 chimeric antibody expression vectors
By heavy chain fragment obtained early period (20H) and light chain segments (21K), heavy chain fragment with Overlap extension PCR and
IgG1Fc constant region (for amino acid sequence as shown in sequence 7 in sequence table, nucleic acid sequence is as shown in sequence 8 in sequence table) splicing exists
Together, light chain segments with Overlap extension PCR and constant region of light chain (amino acid sequence as shown in sequence 5 in sequence table, nucleic acid sequence
As shown in sequence 6 in sequence table) it is stitched together, then recombinate into expression plasmid BP0001, the recombinant expression matter that will be built
Grain transfection CHO cell, expresses overall length humanized antibody.
Concrete operations are (the primer is shown in Table 2) as described below: (1) light chain need to be spliced with Overlap extension PCR and CK constant region
Segment is binned in carrier for expression of eukaryon BP0001 together.Bridge joint PCR is carried out by primer P144, P145 first and obtains light chain
G1 segment, while P146, P147 being utilized to carry out PCR amplification and obtain light chain g2 segment, light chain g1 and g2 segment is passed through into bridge joint PCR
Amplification obtains light chain gene segment, and light chain gene segment is with AvrII enzyme (R0174L, NEB Products) and PacI enzyme
After (R0547L, NEB Products) double digestion, with T4Ligase (M0202L, NEB Products) and same double digestion
BP0001 large fragment is connected, and correct through digestion and sequencing identification, successfully obtains light chain expression vector BP4030 (Fig. 1).(2)
Heavy chain need to be stitched together with Overlap extension PCR and IgG1Fc constant region, then be recombinated into carrier for expression of eukaryon BP0001.First
Bridge joint PCR is carried out by primer P148, P149 and obtains heavy chain g1 segment, while it is light to utilize P150, P151 to carry out PCR amplification acquisition
Heavy chain g1 and g2 segment is obtained light chain gene segment by bridge joint PCR amplification by chain g2 segment, and light chain gene segment is with AvrII
After enzyme (R0174L, NEB Products) and PacI enzyme (R0547L, NEB Products) double digestion, with T4Ligase (M0202L,
NEB Products) it is connected with the BP0001 large fragment of same double digestion, and it is correct through digestion and sequencing identification, it successfully obtains
Heavy chain expression vector BP4031 (Fig. 2)；Same method obtains another heavy chain expression vector BP4032.
Primer needed for 2 PCR system of table
| Primer | Primer base sequences |
| P144 | 5-CGACCTAGGGCCACCATGGACATGAGGGTGCCTGCCCAGCTGCTGGGACTGCTGCTGC-3 |
| P145 | 5-CTGGTTCATCTGGACATCACACCTGGCGCCCCTCAGCCACAGCAGCAGCAGTCCCAGC-3 |
| P146 | 5’-GATGTCCAGATGAACCAG-3’ |
| P147 | 5-CGCTTAATTAACACTCTCCCCTGTTGAAG-3 |
| P148 | 5-CGACCTAGGGCCACCATGGCCTGGATGATGCTGCTGCTGGGACTGCTGGCCTACG-3 |
| P149 | 5-GACTCCACCAGCAGCACTTCGGAGTCCACGCCGCTTCCGTAGGCCAGCAGTCCC-3 |
| P150 | 5’-GAAGTGCTGCTGGTGGAGTC-3’ |
| P151 | 5-CGCTTAATTAATCACTTGCCCAGGCTCAGGCTC-3 |
3, plasmid transfection and cell screening
The above-mentioned successful antibody expression vector of building (BP4030, BP4031 and BP4032) utilizes QIAGEN Plasmid
Midi Kit extracts plasmid, using host cell CHO-S or DG44.By corresponding plasmid stabilisation corotation transfected cho cell, wherein
BP4030 and BP4031, BP4032 and BP4030 corotation.Be transferred to plasmid cell be placed in shaking flask culture (37 DEG C, 5%CO2,
110rpm/min), for recombinant full-lenght antibody-secreting into cells and supernatant, above pureization obtains corresponding overall length IgG antibody
4030/4031 and 4032/4031.Specific purification step is as follows: 1) chromatographic stuffing
Mabselect Sure (GE Products), Superdex 200 (GE Products GE)
2) buffer
Affine equilibration buffer (PBS): 0.2M disodium hydrogen phosphate: 82.5mL/L；0.2M sodium dihydrogen phosphate: 17.5mL/L；
2M sodium chloride: 75mL/L；Ultrapure water is added, mixing is sufficiently stirred, with 1M sodium hydroxide or 1M salt acid for adjusting pH to 7.1-7.3.
Affinity elution buffer: 2.9mL/L acetic anhydride, pH3.4-3.6 is added in NaCl 2.922g/L, anhydrous sodium acetate 0.49g/L.
Affine regeneration buffer: 5.8mL/L acetic anhydride pH3.0.Affine CIP buffer: 1M sodium hydroxide 100mL/L.Gel filtration
It chromatographs solution (PBS): 0.2M disodium hydrogen phosphate: 82.5mL/L；0.2M sodium dihydrogen phosphate: 17.5mL/L；2M sodium chloride: 75mL/
L；Ultrapure water is added, mixing is sufficiently stirred, with 1M sodium hydroxide or 1M salt acid for adjusting pH to 6.8.
3) preparation of samples (clarification filtration) is centrifuged: 5000-6000rpm, 10-15 minutes, Centrifuge A sample.Filtering: centrifugation
Sample supernatant is filtered using H7 (0.45+0.2um) filter afterwards.
4) affinity chromatography
Install AKTA purification system and affinity column (Mabselect or Mabselect Sure).It is rushed with ultrapure water
Wash 2 times of column volumes.With affine 5 column volume of Equilibration buffer wash, until baseline stability, loading.After end of the sample, put down with affine
The buffer that weighs rinses 5~10 column volumes.Column is washed with affinity elution buffer, the main absorption peak at 280nm is collected, washes 1 after afterflow
Column volume.3 column volumes are washed with affine regeneration buffer stream.With affine Equilibration buffer wash to pH neutrality.Use Mabselect
5 column volume of CIP buffer or Mabselect Sure CIP buffer incumbent firms.With affine 3 cylinder of Equilibration buffer wash
Product, until baseline stability.With 3 column volume of ultrapure water, until baseline stability.3 column volumes are rinsed with 20% ethyl alcohol, are saved
Affinity column.
5) gel permeation chromatography
Install 200 gel permeation chromatography column of AKTA explorer purification system and Superdex.Adjust flow velocity 5ml/
Min rinses 2 column volumes with Superdex200 column chromatography equilibrium liquid with 1 column volume of ultrapure water.Adjust flow velocity 2.5ml/
Min takes the direct loading in affine collection peak after adjusting pH.After end of the sample, with the chromatography equilibrium liquid elution of Superdex200 column, receive
Collect purpose peak at 280nm.Continue to rinse 1 column volume.Chromatographic column is saved with 0.01M NaOH.
2 chimeric antibody of embodiment and people PCSK9 binding affinity
The binding affinity of chimeric antibody shown in the present invention and people PCSK9 is measured by biomembrane interference technique.Firstly,
People PCSK9 (R&D company) is subjected to biotin labeling, obtains biotinylated people PCSK9.Again in Otect QKe(Pall
Fortebio) solidified on protein molecular interaction instrument in SA biosensor sensor (Pall company), and in phase
After balancing in buffer, it is transferred to the sample well of the chimeric antibody containing continuous three times dilution (600nM to 0.82nM) respectively
In be combined dissociation reaction.Using Octet software records response signal and calculations incorporated kinetic parameter, it is summarised in table 1,
Data are indicated with Mean ± SD.
The binding kinetics parameter of 1. chimeric antibody of table and PCSK9
3 chimeric antibody biological activity determination of embodiment
1, influence of the chimeric antibody to PCKS9 degradation liver cell LDLR
Human hepatoma HepG2 cell cultivates to logarithmic growth phase, and it is primary to wash cell with PBS, and it is outstanding that cell is made through pancreatin digestion
Liquid and adjust cell density be 1 × 106cells/ml.Inoculating cell is added 200 in 96 well culture plates (100 hole μ l/), edge hole
μ l culture medium.Culture plate is set in 37 DEG C, 5%CO2It is cultivated 24 hours in incubator.Cells and supernatant is removed, every hole is added 100
μ l serum-free cell culture medium continues culture 16 hours；By people PCSK9 (25 μ g/ml) and 100nM, 75nM, 50nM, 25nM,
12.5nM, 6.25nM chimeric antibody preincubate 2h in serum-free h-DMEM culture medium, are added to the cell hole of free serum culture
In, 37 DEG C are placed in, 5%CO2 incubator is incubated for 6 hours, is used for LDLR Immunofluorescence test.
Immunofluorescence test: being rinsed cell 3 times with PBS, and 4% paraformaldehyde is added and fixes, 0.1%triton-X100's
PBST is rinsed 3 times, and LDLR antibody (santa cruz, sc-373830) 4 DEG C of overnight incubations are added；0.1%triton-X100's
PBST is rinsed 3 times, and secondary antibody (green fluorescence label, lifetech, A11001) incubation at room temperature 1h is added, and PBST is rinsed 3 times
Row DAPI is dyed afterwards, is placed under ZEISS laser confocal microscope and is observed, and picture and fluorescence intensity are acquired.
It is jointly processed by HepG2 cell with chimeric antibody provided by the invention and people PCSK9, the results show that two kinds of inosculating antibodies
The LDLR degradation that body can obviously inhibit PCSK9 to mediate, and apparent concentration-dependent relation is presented (see Fig. 1).
2, influence of the chimeric antibody to liver cell intake LDL
Human hepatoma HepG2 cell cultivates to logarithmic growth phase, and it is primary to wash cell with PBS, and it is outstanding that cell is made through pancreatin digestion
Liquid and adjust cell density be 1 × 106cells/ml.Inoculating cell is added 200 in 96 well culture plates (100 hole μ l/), edge hole
μ l culture medium.Culture plate is set in 37 DEG C, 5%CO2In incubator, cultivate 24 hours.Cells and supernatant is removed, every hole is added
100 μ l serum-free cell culture mediums continue culture 16 hours；By people PCSK9 (25 μ g/ml) and 100nM, 75nM, 50nM,
25nM, 12.5nM, 6.25nM chimeric antibody preincubate 2h in serum-free h-DMEM culture medium, are added to the thin of free serum culture
In hilum, 37 DEG C are placed in, 5%CO2 incubator is incubated for 3 hours, is addedLDL (final concentration of 6 μ g/ml) is incubated
3h is educated, supernatant is removed, is rinsed cell 3 times with PBS, is placed on multi-function microplate reader and measures fluorescence intensity, or is being inverted fluorescence microscopy
Microscopic observation and calculating fluorescence intensity.
It is jointly processed by HepG2 cell with chimeric antibody provided by the invention and people PCSK9, the results show that chimeric antibody energy
It enough obviously increases intake of the liver cell to LDL, and apparent concentration-dependent relation is presented (see Fig. 2).This result is from another approach
It confirms that the present invention can obviously inhibit degradation of the PCSK9 to LDLR, promotes LDLR and absorb LDL, and then absorb LDL-C.,
The above result shows that chimeric antibody provided by the invention has good bioactivity.
The ELISA method of 4 chimeric antibody quantitative detection people PCSK9 of embodiment
With the sandwich ELISA assay of anti-human PCSK9 chimeric antibody quantitative detection people PCSK9.Specifically, will with coating buffer
Anti-human PCSK9 chimeric antibody is coated in enzyme mark plate, the hole 200ng/, 4 DEG C of 16~20h of incubation.PBS containing 0.05%Tween-20
After washing 3 times, 2h is closed with the PBS of 3%BSA, 37 DEG C.PCSK9 (the R&D of various concentration is added after being washed 3 times with PBS
Company) 37 DEG C be incubated for 2h.After PBS is washed 3 times, 37 DEG C of the Goat anti-Human PCSK9 antibody (R&D company) of biotin labeling is added
It is incubated for 2h.It is washed 3 times with PBS, adds the streptavidin incubation at room temperature 1h that lotus root is associated with horseradish peroxidase.PBS is washed 3 times
Afterwards, add TMB to carry out chromogenic reaction, and terminated and reacted with 2M sulfuric acid, detect light absorption value at 450nm.The result is that the ELISA method is fixed
Amount detection people PCSK9 deviation in 3.125~200ng/ml concentration range is no more than 20%, and error is no more than 15%.
PCSK9 in 5 chimeric antibody quantitative detection clinical serum sample of embodiment
15 familial hyperlipidemia patients and 10 Healthy People blood are had detected using the sandwich ELISA method of embodiment 1
Clear PCSK9 is horizontal, while having detected serum LDL-C, TG level.15 familial hyperlipidemia patients serum's PCSK9 average waters
It puts down as 116.75 ± 23.04ng/ml；10 Healthy Human Serum PCSK9 average levels are 67.29 ± 29.47ng/ml.15 families
Race's property hyperlipidemia patient serum LDL-C and TG level is respectively 4.95 ± 0.31mmol/L and 3.05 ± 0.65mmol/L；10
Example Healthy Human Serum LDL-C and TG level is respectively 2.52 ± 0.42mmol/L and 1.67 ± 0.23mmol/L.
Claims (3)
1. the Chimeric antibody of specific bond people PCSK9, it is characterised in that the chimeric antibody includes SEQ ID NO:1 institute
Heavy chain variable amino acid sequence shown in the chain variable region amino acid sequence and SEQ ID NO:3 shown, the chimeric antibody
It further include human IgG antibody's constant region, the IgG antibody constant region is human IgG1 or IgG4 constant region.
2. antibody described in claim 1 is in the purposes of the drug of preparation treatment cardiovascular related diseases.
3. antibody described in claim 1 is in preparation clinical diagnosis detection reagent or the purposes of detection kit.
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| CA3009904A1 (en) * | 2015-12-31 | 2017-07-06 | Jiangsu Hengrui Medicine Co., Ltd. | Pcsk9 antibody, antigen-binding fragment thereof, and medicinal application thereof |
| WO2018054241A1 (en) * | 2016-09-20 | 2018-03-29 | Wuxi Biologics (Shanghai) Co., Ltd. | Novel anti-pcsk9 antibodies |
| CN108424457B (en) * | 2017-02-13 | 2021-06-01 | 成都金洛克锶生物技术有限公司 | Preparation and application of PCSK 9-specific antibody and detection kit |
| IT201900000651A1 (en) | 2019-01-16 | 2019-04-16 | Pastore Lucio | Gene transfer technology |
| CN110903397B (en) * | 2019-10-22 | 2022-07-12 | 吉林医药学院 | PCSK9 specific fully human antibody with low-density lipoprotein reduction function and application thereof |
| CN110981962B (en) * | 2019-12-19 | 2022-07-12 | 中国药科大学 | PCSK9 antibody, antigen-binding fragment thereof and application thereof |
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