CN106749663A - Preparation method of the new PD1FAB antibody mabs in DG44 expression systems - Google Patents
Preparation method of the new PD1FAB antibody mabs in DG44 expression systems Download PDFInfo
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Abstract
The present invention relates to preparation method of the new PD1 FAB antibody mabs in DG44 expression systems, including：（a）N-terminal is respectively provided with the gene cloning of amino acid sequence of light chain and the heavy chain of the Fab fragments of signal peptide in building recombinant expression carrier in the expression vector with double-promoter；（b）By in recombinant expression carrier swivel base to the competent cell of gained, screening restructuring bacterial plaque, extract recombinant plasmid, and transfected into DG44 cells, obtain the further amplifying cells of recombinant cell, and Fab albumen is expressed using the cell that cell amplifies culture, the supernatant product containing Fab albumen is collected by centrifugation；（c）Gained cell expression supernatant is concentrated by ultrafiltration first by film bag and cushioning liquid is replaced, then chromatography, you can obtained full human monoclonal antibody PD1 monoclonal antibodies Fab fragments.
Description
Technical field
The present invention relates to preparation method of the new PD1 FAB antibody mabs in DG44 expression systems, belong to biotechnology
Field.
Background technology
PD1 (programmed death 1) antibody mab is famous immune tumor therapeutic antibody global in recent years,
It is the monoclonal antibody drug of the first tumor immunotherapy for obtaining FDA approvals, the breakthrough type for being referred to as treatment of cancer finds, its allusion quotation
The representative of type includes Nivolumab (BMS) and Keytruda (Merk).PD-1 programmed death acceptors are a kind of important being immunized
Suppress molecule, be CD28 superfamily members.PD-1 is main to express in the T cell and B cell of activation, and function is to suppress cell
Activation, this is a kind of normal homeostasis of immune system, because excessive T/B cell-stimulatings can cause autoimmunity disease,
So PD-1 is one amulet of our human bodies.The ability of the escape immune system that tumour cell has, is by it
The programmed death ligand (PD-L1) that surface produces is attached to what is realized on the PD-1 albumen of T cell.Tumour micro-loop in body
Border can induce the T cell of infiltration expression PD-1 molecules high, and the part PD-L1 and PD-L2 of tumour cell meeting expression PD-1 high cause
PD-1 paths sustained activation in tumor microenvironment, T cell function is suppressed and can not find tumour so that it cannot to siberian crabapple
System sends the treatment for needing to attack tumour and killing tumor cell.PD-1 antibody is directed to a kind of antibody proteins of PD-1 so that preceding
Two kinds of albumen can not be combined, and block this path, and the function of T cell is recovered in part, make these cells can continue to kill
Tumour cell.Clinical efficacy on PD-1 antibody, current existing result includes：PD-1 antibody can control 50% cutaneum carcinoma
Cancer progression, cures 10% or so cutaneum carcinoma patient；Also 24% patient with obstinate non-small cell lung cancer can be controlled.When hundred
U.S.A applies the newest clinical test of your treasured and shows that their PD-1 monoclonal antibodies Nivolumab can make 41% patient survive more than 1 year.
Keytruda treatment MPM disease control rates are up to 76%, Keytruda can improve the whole of advanced melanoma
Body survival rate and Progression free survival rate etc..
New PD1 FAB antibody is developed by Shanghai Mei Dixi biological medicines joint-stock company, is belonging to IgG antibody molecule, is
The Fab fragments of Keytrud.New PD1 FAB antibody is the dimer of human monoclonal antibodies heavy chain and light chain through disulfide-bonded,
Specifically can be combined with PD1 so as to block PD1/PD-L1 signal paths.
PD1 FAB antibody is the antigen conjugates of IgG molecules, be by heavy chain and light chain by disulfide-bonded formed it is different
Dimerization is fit, because the integrality structure and the low advantage of immunogenicity that its relative molecular mass is small, have height determination turn into IgG classes
Antibody producing and the first-selection of transformation.DG44 cell lines, are dfhr defective Chinese hamster ovary celIs systems, can high level expression foreign gene；Greatly
The recombinant protein of majority expression is solvable；Processing modification after being translated：Including glycosylation, phosphorylation, acylation, signal peptide
Excision etc.；It is easier to isolate and purify；DG44 cells suspension growth easily amplifies culture, is conducive to expressing recombinant protein on a large scale.It is
A kind of conventional therapeutic antibodies expression cell system.But there is presently no by DG44 cells be used for express PD1 FAB antibody
Report.
The content of the invention
Recombinated it is an object of the invention to provide coexpression is secreted in a kind of DG44 mammalian cell expression systems
PD1 FAB antibody.The present inventor it has been investigated that, expressed as host by using DG44 cells, can obtain with life
The restructuring PD1 FAB antibody of thing activity.
Here, the present invention provides a kind of brand-new PD1 FAB antibody (is also known as PD1 Fab fragments, PD1 monoclonal antibodies herein
Fab fragments etc.) preparation method, including：
A () N-terminal is respectively provided with the heavy chain (Heavy chain, Hc) of signal peptide and the ammonia of light chain (Light chain, Lc)
The gene cloning of base acid sequence is in expression vector (such as pOptiVEC with double-promoterTM) interior structure recombinant expression carrier；
B (), by recombinant expression carrier swivel base to the competent cell of gained, screening restructuring bacterial plaque extracts recombinant plasmid, and will
In its transfection to mammalian cell, by limiting dilution assay, foundation surely turns cell line, protein-contg supernatant is collected by centrifugation and produces
Thing；
C first by film bag be concentrated by ultrafiltration gained mammalian cell expression supernatant and cushioning liquid is replaced by (), then chromatogram
Post is purified, you can obtain brand-new PD1 FAB antibody.
The present invention divides the heavy chain of the PD1 FAB antibody with signal peptide with light chain with technological means such as genetic engineerings
POptiVEC is not cloned into itTM) in, recombinant plasmid is obtained, will be obtained in recombinant plasmid swivel base to competent cell (such as Top10)
Recombinant plasmid, transfection mammalian cell DG44 obtains the cell line of stabilization expression PD1 FAB antibody, secreting, expressing purpose egg
In vain, the supernatant product containing destination protein is collected by centrifugation.Chromatography is carried out again, obtains the PD1 FAB antibody of bioactivity
Albumen.Brand-new PD1 FAB antibody is obtained through the inventive method.
In the present invention, from DG44 fibrocyte expression vectors pOptiVECTMExpression, can accommodate the Insert Fragment of macromolecular, table
Up to various exogenous genes and can efficiently and mass expressing external gene.Coexpression can be secreted by adding outer secreting signal peptide
PD1 FAB antibody proteins using extracellular oxidation environment, can instruct the heavy chain of PD1 FAB antibody and light in cell conditioned medium
The formation of interchain disulfide bond.
Preferably, in step (a), the recombinant expression carrier is pOptiVECTM, including：Two promoters, duplications
Son, polyclone enzyme enzyme site and two terminators.
Preferably, in step (a), the signal peptide of the N-terminal of the Lc and Hc of PD1 FAB antibody is outer secreting signal peptide Ac
NPV gp64.The heavy chain of PD1 FAB antibody and the promoter of light chain can be all polyhedrosis gene promoter.
Preferably, in step (b), the competence is Top1.It is preferred that swivel base bacterial plaque screening technique is blue hickie sieve
Select method.
Preferably, in step (b), transfection reagent can be liposome cellfection, be obtained surely by limiting dilution assay
The cell line of fixed expression high.
Preferably, in step (b), the recombinant cell lines P0 expands P1, will 200 μ L P0 add 100mL cells close
Spend is 2 × 106It is centrifuged in the DG44 cells of cells/ml, after 37 DEG C of shaking table culture 96h and obtains cell line P1.
Preferably, it is ESF culture mediums that cultured cells is amplified in step (b) and culture medium used is expressed.
Preferably, in step (c), the purifying includes successively：It is molten that buffering is concentrated by ultrafiltration and replaced using film bag
Liquid；And affinity chromatography and molecular sieve medium chromatography.
Preferably, in step (c), the affinity chromatography medium of the affinity column chromatography is Protein A.
Preferably, in step (c), the sieve chromatography medium of the molecular sieve medium chromatography is Superdex 75.
Brief description of the drawings
Fig. 1 is PD1 FAB antibody fragment recombinant expression carrier collection of illustrative plates；
Fig. 2 is the PD1 FAB antibody SDS-PAGEs after Protein A affinity purifications；Figure A therein is in non-reduced bar
Electrophoresis acquired results under part, figure B is electrophoresis acquired results under reducing condition；PD1 FAB are IgG4 antigen binding domains Hc and Lc bis-
Aggressiveness, is two electrophoretic bands of 23kD under reducing condition, there is single electrophoretic band under non reducing conditions at 47kD；
Fig. 3 is sieve chromatography PD1 FAB SDS-PAGEs after purification, and figure A therein is the electricity under non reducing conditions
Swimming acquired results, figure B is electrophoresis acquired results under reducing condition；
Fig. 4 is purified and PD1 FAB SDS-PAGEs after concentrating for sieve chromatography, shows the PD1 FAB antibody for obtaining
Fragment has higher degree.
Specific embodiment
The present invention is further illustrated below in conjunction with accompanying drawing and following implementation methods, it should be appreciated that accompanying drawing and following implementation methods
The present invention is merely to illustrate, is not intended to limit the present invention.
The present invention uses the means such as genetic engineering, using pOptiVECTMAs expression vector, by the use of DG44 cells as place
It is main, express PD1 FAB antibody fragments.In one example, preparation method of the invention includes：Ac NPV will be respectively provided with
The heavy chain and light chain of the PD1 FAB antibody of gp64 signal peptides are cloned into carrier pOptiVECTMIn, recombinant expression carrier is built, will
Recombinant plasmid swivel base is to acquisition recombinant plasmid dna in competent cell Top10 and transfects into cell, by limiting dilution assay,
Expression monoclonal cell strain high is obtained, PD1 FAB destination proteins are expressed, centrifugation obtains the supernatant product containing destination protein；To institute
The albumen for obtaining carries out chromatography, obtains PD1 FAB albumen.Hereinafter, preparation method of the invention is exemplarily illustrated.
(1) structure of recombinant expression carrier
The heavy chain of the PD1 FAB fragments with signal peptide and the gene of light chain are respectively synthesized, are cloned in double-promoter
In pOptiVECTM carriers, PD1 FAB antibody fragments recombinant expression carrier (PD1 FAB in pOptiVEC are builtTM)。
Preferably, the N-terminal in Hc and Lc respectively adds an outer secreting signal peptide Ac NPV gp64.
In one example, signal peptide Ac NPV gp64 are added before Lc and forms AcNPV gp64+Lc in
pOptiVECTM.Preferably, it is inserted in after first promoter using 5'BamHI and 3'EcoRI.
In another example, signal peptide Ac NPV gp64 formation AcNPV gp64+Hc in were added before Hc
pOptiVECTM.Preferably, it is inserted in after second promoter using 5'XhoI and 3'HindIII.
Constructed PD1 FAB antibody fragment recombinant expression carrier collection of illustrative plates is as shown in Figure 1.As shown in Figure 1, the recombination expression
Carrier includes：Two cellular promoters, replicon, polyclone enzyme enzyme site and two terminators.
(2) expression of the PD1 FAB fragments in cell
The preparation of recombinant plasmid：By constructed PD1 FAB antibody fragment recombinant expression carrier swivel bases to competent cell
(TOP10) in, screening restructuring bacterial plaque extracts recombinant plasmid.
Transfected Recombinant Plasmid DG44 cells, transfection reagent can be liposome cellfection.By limiting dilution assay, use
Nutrient solution is diluted, and makes cell concentration for 50~60/mL, and 0.1mL (five or six cells/wells) is added per hole in 96 well culture plates.
2 rows are inoculated with, remaining cell suspension nutrient solution makees doubling dilution, inoculates 2 rows, and so on, until making every hole contain 0.5~1
Individual cell.After 7~10d of culture, the positive hole of single clonal growth is selected to be cloned again.Generally require and so repeat 3
~5 times, when up to 100% positive porosity, to ensure antibody as produced by single clone.Obtain PD1 FAB-P0 generations thin
Born of the same parents system.The P0 that transfection is obtained obtains P1 for the further amplifying cells of cell.In one example, amplification method is：By 200 μ L weights
It is 2 × 10 that group P0 cells add 100mL cell densities6In the DG44 cells of cells/ml, it is centrifuged after 37 DEG C of shaking table culture 96h and is obtained
Obtain cell P1.PD1 FAB albumen is expressed using P1 cell lines.
(3) purifying of PD1 FAB
PD1 FAB albumen is expressed as the outer secreting, expressing of solubility, and expression culture medium used can be ESF culture mediums, and cell expands
Cell suspension is collected when increasing cell culture to percentage of cells 80% or so, 10min is centrifuged at 4 DEG C of centrifuge 3000g, collected
Supernatant solution, supernatant is concentrated and replaced buffer solution using the cross-flow ultrafiltration film bags of Vivaflow 200, facilitates follow-up
Isolation and purification.Then chromatography is carried out, the PD1 FAB fragments of high-purity are obtained.Preferably, affinity column layer is passed sequentially through
Analysis and molecular sieve medium chromatography are purified.In one example, the affinity chromatography medium of affinity column chromatography is Protein A.
In another example, the sieve chromatography medium of molecular sieve medium chromatography is Superdex 75.In purifying, can make
With SDS-PAGE electrophoretic analysis purity of protein.30kD Milipore super filter tube protein concentrates can be used.
(analysis and detection)
SDS-PAGE electrophoretic analysis destination protein PD1 FAB fragments can be used.Fig. 2,3 are shown respectively through affinity column chromatography, divide
Son sieve medium chromatographs the SDS-PAGE of obtained albumen, is used to characterize the purity of albumen.Figure A in the two width figure is equal
It is non reducing conditions, figure B is reducing condition.As shown in Figure 2, PD1 FAB are the dimer of IgG4 antigen binding domains Hc and Lc,
There are two electrophoretic bands under reducing condition at 23kD, respectively Hc and Lc, has under non reducing conditions at 47kD from top to bottom
Single electrophoretic band is PD1 FAB fragments.Fig. 4 show sieve chromatography purify and PD1 FAB albumen after concentrating SDS-
PAGE electrophoretograms, the wherein sample of swimming lane 1 are treatment under non reducing conditions, and swimming lane 2 is treatment under reducing condition, is contracted as shown in Figure 4
The purity of PD1 FAB antibody fragments afterwards is more than 98%.
The present invention has the advantage that compared with prior art：
(1) the PD1 FAB fragments molecules amounts prepared by the present invention are small, therefore the penetration power during tumour target site is reached
Greatly；
(2) the PD1 FAB fragments prepared by the present invention remain high specific and high-affinity of the whole antibody to PD1 FAB, and
And its half-life period is long compared with similar drugs, it is possible to reduce the access times of medicine；
(3) recombinant protein of DG44 system expressions has complete biological function, can correctly fold and be formed PD1 FAB molecules
Between important disulfide bond, expression product is in structure and functionally close to native protein；
(4) the processing modification after being translated：Including glycosylation, phosphorylation, acylation, signal peptide excision etc.；
(5) compared with other eukaryotic expression systems, DG44 expression systems can be high with efficiently expressing exogenous gene, expression quantity；
(6) Insert Fragment of macromolecular can be accommodated, big genetic fragment can be packed and various external source bases can be expressed in cell
Cause；
(7) in purge process, purified using affinity chromatography-molecular sieve chromatography and obtain antibody protein of the purity more than 98%, be
It is a kind of simple to operate, low cost, the antibody fragment purification preparation method of high production；
(8) the invention provides a kind of preparation method of PD1 FAB antibody fragments, to obtain high-quality monoclonal antibody PD1
FAB fragments are laid a good foundation.
Embodiment is enumerated further below to describe the present invention in detail.It will similarly be understood that following examples are served only for this
Invention is further described, it is impossible to be interpreted as limiting the scope of the invention, those skilled in the art is according to this hair
Some nonessential modifications and adaptations that bright the above is made belong to protection scope of the present invention.Following examples are specific
Technological parameter etc. is also only that an example in OK range, i.e. those skilled in the art can be done properly by the explanation of this paper
In the range of select, and do not really want to be defined in the concrete numerical value of hereafter example.The examination of unreceipted actual conditions in the following example
Proved recipe method, generally according to normal condition, or according to the condition proposed by manufacturer.
(1) structure of recombinant plasmid
Amplification gene Lc first, adds signal peptide AcNPV gp64 (autographa california nuclear polyhedrosis virus) by gene
AcNPV gp64+Lc are cloned into carrier pOptiVECTM (Life technology), build plasmid AcNPV gp64+Lc in
pOptiVECTM.Then base is expanded from template plasmid PD1 FAB in pETDuet1 (Shanghai Medicilon Inc.)
Because of Hc, signal peptide AcNPVgp64,3 ' terminator codon TAA are added, Gene A cNPV gp64+Hc are cloned into carrier
POptiVECTM, builds plasmid AcNPVgp64+Hc in pOptiVECTM.Finally from template plasmid Ac NPV gp64+Hc in
Amplification gene PH promoter+Ac NPV gp64+Hc in pOptiVECTM, using the method for restructuring by gene PH
Promoter+Ac NPV gp64+Hc are cloned into carrier AcNPV gp64+Lc in pOptiVECTM, build plasmid Ac NPV
gp64+Lc and Ac NPV gp64+Hc in pOptiVECTM。
(2) expression of the PD1 FAB in DG44 cells
DG44 cells (Life technology) at 27 DEG C, Shaking culture under conditions of 90rpm.When DG44 cell concentrations reach
2.0×106During cells/ml, the ratio with infection multiplicity (MOI) as 1pfu/cell adds P1, and for cell, (i.e. every liter cell adds
Enter the P1 cells of 20ml), in cell amplification is carried out in 27 DEG C of incubators, culture medium is ESF culture mediums (Life
technology).PD1 FAB albumen is expressed as the outer secreting, expressing of solubility, cell amplification to percentage of cells 80% or so
When collect cell suspension, 4 DEG C of centrifugation 10min of centrifuge 3000g collect cell conditioned medium.
(3) concentration of PD1 FAB expression supernatant and buffer exchange
PD1 FAB albumen is expressed as the outer secreting, expressing of solubility, and the supernatant solution being collected by centrifugation is cut using Vivaflow 200
Buffer solution (50mmol/L Tris-HCl pH7.2,150mmol/L NaCl, 10% are concentrated and replaced to stream milipore filter bag
Glycerol), follow-up isolation and purification is facilitated.
(4) chromatography
First step Protein A affinity chromatographys
1) chromatographic column：Affinity chromatography medium：Protein A are affine filler (GE)；
2) preparation of Protein A affinity columns
3) sample treatment：The supernatant solution centrifuging and taking supernatant of concentration and displacement cushioning liquid will be completed；
4) buffer solution configuration：Buffer A：50mmol/L Tris-HCl pH7.2,150mmol/L NaCl, 10%Glycerol,
Buffer B：0.1mol/L Glycine,pH 2.5；
5) purge process：With buffer A pre-balance filler, pillar is balanced with buffer A after sample loading, then use buffer B
Wash-out, Ep pipes are collected and neutralize the PD1 FAB albumen under wash-out to pH 7.0 or so；
6) result：Target protein peak is collected, protein concentration is surveyed and is calculated the rate of recovery, SDS-PAGE analyzing proteins purity (Fig. 2).As a result
Show that the purity of protein is more than 90%.
Second step sieve chromatography
1) chromatographic column：HiLoad 16/60Superdex 75prep grade, GE companies of the U.S.；
2) sample treatment：The target protein eluted by Protein A affinity chromatographys is the ultrafiltration of 30KD using molecular cut off
Concentration tube is concentrated into 2-4ml；
3) buffer solution configuration：Buffer solution is 50mmol/L Tris-HCl pH7.0,150mmol/L NaCl, 10%Glycerol；
4) purge process：With buffer solution pre-balance molecular sieve, loading is eluted with 1ml/min；
5) result：Target protein peak is collected, sample carries out SDS-PAGE electrophoresis (Fig. 3).Result shows that the purity of protein is more than
98%.
Finally, purified product is concentrated by ultrafiltration using Millipore ultrafiltration systems, molecular cut off is fine for the regeneration of 30KD
The plain film bag of dimension carries out desalination and concentration to protein solution after purification, and whole purifying combination is reasonable, and simple to operate, technique is steady
It is fixed.SDS-PAGE analyzing proteins purity (Fig. 4).
Claims (9)
1. a kind of method of use DG44 cell expression systems Prepare restructuring PD1 monoclonal antibody Fab fragments, it is characterised in that including：
（a）N-terminal is respectively provided with the gene cloning of light chain and the amino acid sequence of heavy chain of the Fab fragments of signal peptide in double
Recombinant expression carrier is built in the expression vector of promoter；
（b）By in recombinant expression carrier swivel base to the competent cell of gained, screening restructuring bacterial plaque extracts recombinant plasmid, and will
In its transfection to DG44 cell, the further amplifying cells of recombinant cell are obtained, and express using the cell that cell amplifies culture
Fab albumen, is collected by centrifugation the supernatant product containing Fab albumen；
（c）Gained cell expression supernatant is concentrated by ultrafiltration first by film bag and cushioning liquid is replaced, then chromatographic column is pure
Change, you can obtain full human monoclonal antibody PD1 monoclonal antibodies Fab fragments.
2. preparation method according to claim 1, it is characterised in that in step（a）In, the heavy chain and light chain of Fab fragments
The signal peptide of N-terminal be all outer secreting signal peptide Ac NPV gp64.
3. preparation method according to claim 1 and 2, it is characterised in that in step（a）In, the heavy chain of Fab fragments and light
The promoter of chain is all polyhedrosis gene promoter.
4. preparation method according to any one of claim 1 to 3, it is characterised in that in step（a）In, the restructuring
Expression vector is pOptiVECTM, including：Two promoters, replicon, polyclone enzyme enzyme site and two terminators.
5. preparation method according to any one of claim 1 to 4, it is characterised in that in step（b）In, the impression
State is Top10.
6. preparation method according to any one of claim 1 to 5, it is characterised in that transfection reagent is liposome
cellfection。
7. preparation method according to any one of claim 1 to 6, it is characterised in that in step（b）In, the restructuring
Cell P0 amplifying cells P1, will 200 μ L cells P0 add 100 mL cell densities be 2 × 106The DG44 of cells/ml is thin
It is centrifuged in born of the same parents, after 37 DEG C of h of shaking table culture 96 and obtains cell P1.
8. preparation method according to any one of claim 1 to 7, it is characterised in that in step（b）In, amplify culture
Cell simultaneously expresses culture medium used for ESF culture mediums.
9. preparation method according to any one of claim 1 to 8, it is characterised in that in step（c）In, the purifying
Including：Film bag is concentrated by ultrafiltration and is replaced cushioning liquid；And affinity chromatography and molecular sieve medium chromatography；It is wherein described affine
The affinity media of column chromatography is Protein A；The sieve chromatography medium of the molecular sieve medium chromatography is SuperdexTM 75。
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| CN109988238A (en) * | 2017-12-29 | 2019-07-09 | 上海美迪西生物医药股份有限公司 | Preparation method of the novel PD1 single-chain antibody in Escherichia coli |
| CN109988238B (en) * | 2017-12-29 | 2020-12-25 | 上海美迪西生物医药股份有限公司 | Preparation method of PD1 single-chain antibody in escherichia coli |
| WO2020201442A1 (en) | 2019-04-03 | 2020-10-08 | Orega Biotech | Combination therapies based on pd1 and il-17b inhibitors |
| WO2021170750A1 (en) | 2020-02-28 | 2021-09-02 | Orega Biotech | Combination therapies based on ctla4 and il-17b inhibitors |
| EP4257609A1 (en) | 2022-04-08 | 2023-10-11 | iOmx Therapeutics AG | Combination therapies based on pd-1 inhibitors and sik3 inhibitors |
| WO2023194622A1 (en) | 2022-04-08 | 2023-10-12 | Iomx Therapeutics Ag | Combination therapies based on pd-1 inhibitors and sik3 inhibitors |
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