CN104818295A - Method for preparing and screening cell line expressing bispecific antibody - Google Patents
Method for preparing and screening cell line expressing bispecific antibody Download PDFInfo
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Abstract
The present invention provides a method for preparing and screening a cell line expressing bispecific antibody. The method comprises: constructing two plasmids expressing bispecific antibody, wherein the two plasmids contain double promoters and respectively express different fluorescent proteins; and carrying out transformation, culture and extraction on the two recombinant plasmids, co-transfecting the two recombinant plasmids into host cells, screening the positive monoclonal cell line expressing the double fluorescence, and evaluating the yield and the stability according to the fluorescence intensity. With the method of the present invention, the high yield cell line can be conveniently and rapidly screened in advance, the test period can be shortened, the manpower and material resource investment can be reduced, and the accuracy is high.
Description
Technical field
The present invention relates to antibody preparation field, particularly relate to a kind of method prepared and screen expression bi-specific antibody cell strain.
Background technology
Bi-specific antibody (bispecific antibody, hetero conjugate antibody) is that a class has bifunctional antibody molecule, and the Fab section in two valency antibody has not homospecificity, can from different ligand bindings.The bi-specific antibody of antitumor and anti-immunologically competent cell CDl6 or CD3, not only have and activate NK cell or T cell effect, and can be played a role by antineoplastic Fab section specific binding tumour cell, improve local NK cell or T cell concentration, reinforcing effect molecule killing tumor cells ability.Also prepare immune cell factor (immunocytokine) by antibody and some cytokine fusion, strengthen cytokine concentration near tumour cell, excitating organism immunologic function, effectively killing tumor cells, reduce toxic side effect.In addition, also can apply the embedding and property antibody of the antitumor monoclonal antibody of humanized or humanized Fc section and mouse Fab section, overcome murine antibody immunogenicity, strengthen antibody directed cellular toxic action, reach killing tumor cells effect.Contain the Fab section of two kinds of different identification specificity antigens in bi-specific antibody, by specific combination tumour antigen simultaneously in conjunction with different effect cell and molecule, reach the effect of effective killing tumor cells.
At present, Chinese hamster ovary cell (CHO (Cricetulus griseus, hamster, Chinese, ovary) be widely used in production antibody, the cell strain of usual high yield is for sulfone (methionine sulfoximine on the one hand by methotrexate (methotrexate, MTX) or methionine(Met) imino-, MSX) carry out pressurization amplification to obtain, also have a lot of report to obtain high yielding cell sarain by genetically engineered on the other hand.Many companies strengthen the integration efficiency of foreign gene by the plasmid building high transfection efficiency as introduced lentiviral vectors (lentiviralvectors (LVs)), thus improve antibody production or by increasing enhanser and separaant strengthens the vigor of promotor and then finds high yielding cell sarain; Also some company improvement rotaring transfecting mode, as adopted Ca
3(PO4)
2the transfection means such as reagent, PEI reagent or electroporation improve transfection efficiency etc., after improve transfection efficiency, therefrom filter out and find out stable high yielding cell sarain by above method, and the amplification entering downstream is produced, and still needs the process that very long.From traditional experiment flow, this process needs 6 to 12 months just can obtain stable cell strain system usually, and needs to consume a large amount of manpower and materials, finally also not necessarily can obtain desirable high yielding cell sarain.Therefore in scale operation biological products, except building the plasmid vector of good high expression level, improve outside the approach such as transfection efficiency, also need to find one conveniently can filter out high yielding cell sarain in advance, and the method for later stage monitoring cell strain stability, can not only shorten the test period like this, and can reduce the input of manpower and materials, this is also that to prepare bi-specific antibody successfully crucial.
At present, lack a set of perfect can early screening, qualification stably express bi-specific antibody cell strain method; Most of company is detected by the bag of external source and is screened High producing clones cell strain, but can not reflect the expression status of clonal cell line completely truly.
Summary of the invention
Therefore, express bi-specific antibody cell strain stability approach length consuming time to solve at present screening, qualification process is loaded down with trivial details, and the not high weak point of accuracy, the invention provides a kind of method that bi-specific antibody cell strain is expressed in new preparation and screening.
Key of the present invention is that used plasmid is for double-promoter plasmid, and introduces fluorescin as indicating label.The one class homologous protein of fluorescin family to be the molecular weight found from hydrozoa and alcyonarian animal be 20-30kD, mainly comprise green fluorescent protein and its some mutant, as blue fluorescent protein, yellow fluorescence protein, bluish-green fluorescin etc., the red fluorescent protein found from coral in addition.They have the features such as automatic luminous, easily detection, and in biology, they are usually as reporter gene.
Internal ribosomal entry site sequence (internal ribosome entry site, IRES), it is one section of nucleotide sequence, its existence can make that protein translation is initial does not rely on 5 ' cap structure, thus makes directly to become possibility from initiation of translation in the middle of messenger RNA(mRNA) (mRNA).It can work in coordination with coexpression two goal gene in same carrier, without the need to worrying issuable problem in the efficiency of cotransfection and other cotransfection processes.When reporter gene and goal gene work in coordination with coexpression, it is minimum on the bioactive impact of target protein.Commonly use and be implemented in the middle of eukaryote bicistronic mRNA (bicistronic mRNA), thus, first albumen is usually by 5 ' cap sequence initiation of translation, and second albumen then relies on IRES initiation of translation.The expression of two albumen before and after IRES is normally proportional, therefore can reflect the expression of another one albumen according to the expression of one of them reporter gene.
The present invention may be used for screening and expresses bi-specific antibody Chinese hamster ovary celI system.Wherein, bi-specific antibody comprises two structural units: unit one and unit two.Unit one has two kinds of forms, one is that two polypeptide such as heavy chain light chain match, it two is a polypeptide as variable region fragment strand (ScFv, single chainvariable fragments) or ScFv and heavy chain constant domain albumen, with antigen one specific combination; Unit two has the two kind forms identical with unit one, but unit two can be identical with the form of unit one, also can be different.Each cell formation is in a double promoter expression vector, if two polypeptide forms, then fluorescin utilizes internal ribosomal entry site sequence to be connected to the C end of a polypeptide; If a polypeptide form, then fluorescin is directly inserted into another promotor multicloning sites downstream place (multiple cloning site, (MCS)) of carrier.The present invention by transforming in the double-promoter double-mass model system (plasmid A and plasmid B, is dual-promoter vector) expressing bi-specific antibody.Wherein, plasmid A (Fig. 1) expresses unit A, containing a polypeptide, the cDNA of fluorescin A (FPA) is connected in the idle promotor multicloning sites downstream of the plasmid A expressing unit A, plasmid B (Fig. 3) expresses unit B, containing two polypeptide, the cDNA of internal ribosomal entry site sequence, fluorescent protein B (FPB) is connected into successively after arbitrary polypeptide in the plasmid B expressing unit B; In plasmid A and plasmid B cotransfection mammalian cell after coexpression, by the fluorescing matter of flow cytometry analysis cell, judge in cell, whether these two dual anti-unit all express, and by the fluorescent signal that direct-detection is endogenous, screen the cell strain of high yield bi-specific antibody according to fluorescence signal intensity.
The method of bi-specific antibody cell strain is expressed in preparation provided by the invention and screening, and it comprises the following steps:
Step 1: construction expression bi-specific antibody two plasmids, two plasmids all contain double-promoter, and the albumen of one of them plasmid expression is one or two polypeptide of specific combination tumour antigen; The albumen of another plasmid expression is one or two polypeptide of specific combination immunologically competent cell antigen; Described two plasmids containing double-promoter also express a kind of different fluorescin respectively; If the albumen of plasmid expression is two polypeptide forms, then fluorescin utilizes internal ribosomal entry site sequence to be connected to the C end of a polypeptide, if a polypeptide form, then fluorescin is directly inserted into another promotor multicloning sites downstream of carrier;
Step 2: obtain in step 1 two kinds of recombinant plasmids are carried out transforming, cultivate and extracting;
Step 3: by two kinds of recombinant plasmid cotransfections of extraction in step 2 in host cell;
Step 4: the positive monoclonal cell strain of two fluorescence is expressed in screening.
In one embodiment, be the structure selecting expression vector in above-mentioned steps 1, construction unit A-FPA respectively, unit B-IRES-FPB expression vector.According to unit A, B, IRES, FPA, multiple clone site design primer in FPB gene order and carrier, after wherein this fragment gene archaeal dna polymerase of FPA being increased out, cut glue to reclaim, digested plasmid A and FPA reclaims product respectively, FPA is inserted in plasmid A, the new plasmid obtained thus is designated as plasmid ANF (Fig. 2), after unit B-IRES – FPB carries out twice PCR respectively, cut glue to reclaim, carry out over-lap PCR subsequently, after cutting glue recovery, enzyme cut unit B-IRES-FPB fragment respectively, with plasmid B, namely the unit B in original plasmid is replaced with unit B-IRES-FPB, the novel plasmid obtained thus is designated as plasmid BIF (Fig. 4).
In one embodiment, if the albumen of described plasmid expression is a polypeptide, then on plasmid, a promotor is used for initiation transcription protein unit, and another promotor is used for initiation transcription fluorescin; If the albumen of plasmid expression is unit is two polypeptide, then 3 ' of the DNA encoding sequence of a polypeptide on plasmid to connect the DNA encoding sequence of fluorescin by IRES.
In one embodiment, the plasmid that axenic purification extracts also is comprised in step 2 above.By two kinds of recombinant plasmids transformation of E. coli respectively, picking mono-clonal bacterium colony carries out mass propgation, utilizes and extracts plasmid without intracellular toxin test kit, detects plasmid concentration and purity.Institute's upgrading grain is carried out axenic purification, and carries out Sterility testing.
In one embodiment, in above-mentioned steps 3, interpolation screening pressure is also comprised.In step 3 by plasmid ANF and plasmid BIF cotransfection in mammalian cell, within after transfection two days, add corresponding screening pressure.Screening pressure can be tetracycline, Totomycin or methotrexate.
In one embodiment, screen with flow cytometer or fluorescent microscope in above-mentioned steps 4.After cultivating 2 weeks, screening high yielding cell sarain, expresses positive monoclonal cell with flow cytometer or fluorescent microscope sorting.
In one embodiment, also comprise the positive monoclonal cell strain after by screening and carry out enlarged culturing, detect its fluorescence intensity with flow cytometer or fluorescent microscope.Monoclonal cell after sorting is cultivated, after enlarged culturing, detects its fluorescence intensity with flow cytometer or fluorescent microscope, detect its antibody production with ELISA, contact more therebetween.
In one embodiment, described host cell is mammalian cell.Mammalian cell can be Chinese hamster ovary cell.
In one embodiment, described fluorescin correctly can indicate protein expression situation after transfection, can be green fluorescent protein, red fluorescent protein, yellow fluorescence protein, orange fluorescent protein or blue fluorescent protein etc.
In one embodiment, according to the expression bi-specific antibody cell strain that aforesaid method preparation and screening obtain.
In one embodiment, the carrier that plasmid ANF uses is
pCHO 1.0Expression Vector (is called for short pCHO, purchased from Life technologies, article No. A13696-01), but need to transform, wherein will replace with Totomycin (Hygromycin) resistant gene containing puromycin resistance gene, improved carrier called after HpCHO; The carrier that plasmid BIF uses is the pCHO do not transformed, and it is with tetracycline (Puromycin) and methotrexate (MTX) resistant gene.
In one embodiment, also comprise the positive monoclonal cell strain after to screening and utilize flow cytometry to carry out Rapid Stability assessment, within 10 generations, determine the stability of monoclonal cell strain.
In one embodiment, according to the expression bi-specific antibody cell strain that preceding method obtains, in expression product, double antibody relative content (content of the relatively all antibody of double antibody), can be assessed by the average fluorescent strength proportionality of calculating two kinds of fluorescence such as GFP/RFP ratio.
In one embodiment, according to the expression double antibody cell strain that preceding method obtains, the stability of its expression level can be assessed by the flow cyctometry analysis of cells fluorescence intensity such as histogram situation that distributes.
Accompanying drawing explanation
In order to be illustrated more clearly in the technical scheme in the embodiment of the present application, be briefly described to the accompanying drawing used required in embodiment below, apparently, the accompanying drawing that the following describes is only some embodiments recorded in the application, those of ordinary skill in the art are come, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to these accompanying drawings.
Fig. 1 is the expression vector plasmid A structural pattern figure of strand unit.
Fig. 2 is strand unit and fluorescin A (FPA) co-expression carrier plasmid ANF structural pattern figure.
Fig. 3 is the expression vector plasmid B structural pattern figure of unit price unit.
Fig. 4 is unit price unit and fluorescent protein B (FPB) co-expression carrier plasmid BIF structural pattern figure.
Fig. 5 is 48 hours fluorescent detection figure after cotransfection.
Fig. 6 is flow cytometer showed cell strain fluorescing matter figure, and 6A is double negative cells, 6B is single fluorescence (GFP) positive cell and 6C is two fluorescencepositive cell.
The correlation analysis figure of Fig. 7 antibody production and cell average fluorescent strength, 7A are the dependency figure that the dependency figure of GFP average fluorescent strength and antibody production, Fig. 7 B analyzes RFP average fluorescent strength and antibody production.
The antibody purity that Fig. 8 statistical study Western blot detects and streaming fluorescence intensity ratio figure, Fig. 8 A is Western blot figure, swimming lane 1-7 is corresponding is respectively that clone 1-7 expresses supernatant, Fig. 8 B is the dependency between fluorescence intensity ratio and double antibody relative content, fluorescence intensity ratio=GFP average fluorescent strength/RFP average fluorescent strength.
The flow cyctometry histogram of the different clonal cell line of Fig. 9.
The continuous 30 generation yield assessment figure of the different clonal cell line of Figure 10.
Embodiment
One, experiment material
The bi-specific antibody of this experiment is unit price single chain bispecific antibody (MSBODY (monomer andScFv bispecific antibody)), MSBODY is prepared by Wuhan You Zhiyou Biology Pharmacy Co., Ltd method disclosed in PCT/CN2012/084982, and can buy from the said firm.MSBODY is made up of two unit: a unit, for have specific light-heavy chain pair for tumour cell or microorganism, is called unit price unit; Another unit is fusogenic peptide, and described fusogenic peptide comprises single chain variable fragment (scFv) and has the Fc fragment of CH2 structural domain and CH3 structural domain, and wherein said fusogenic peptide has specificity for immunocyte, is called strand unit.Wherein, the constant region of light chain (CL) of unit price unit is completely the same with human IgG antibody's constant region of light chain.The tumor targets antigen that unit price unit specific binding is different or microorganism target antigen, as Her2, EGFR, EpCAM, CD19 and CD20 etc.; The surface antigen of strand unit specific binding immunocyte, as CD3, CD16, CD19, CD28 and CD64 etc.
Green fluorescent protein (GFP) (originate: GenBank access no.U55762), red fluorescent protein (RFP) (originate: GenBank access no.HM771696.1), internal ribosomal entry site sequence (IRES) (originate: GenBank access no.KC710231.1) these gene fragments all give the synthesis of Jin Wei intelligence biotech firm by sequence by sequence by sequence.Other experiment materials have: intestinal bacteria Trans10 is (purchased from Quan Shijin, article No. CD101-02), T4DNA ligase enzyme (NEB, article No. M0202S), PyrobestDNA polysaccharase (Takara, article No. DR005A), restriction enzyme A vrII (NEB, article No. R0174L), restriction enzyme BstZ17I (NEB, article No. R0594L), restriction enzyme EcoRV-HF (NEB, article No. R3195L) and restriction enzyme PacI (NEB, article No. R0547L); Kantlex (Amersco, article No. 0408-10G); Plasmid extraction kit (purchased from Tiangen, article No. DP104), DNA fragmentation glue reclaim test kit (Tiangen, article No. DP210); Carrier for expression of eukaryon
pCHO 1.0Expression Vector (being called for short pCHO), mammalian cell strain CHO-S, culture medium C DForti-CHO, substratum OptiPRO
tMsFM and transfection reagent FreeStyle
tMmAX Reagent is all from test kit
kit (Life technologies, article No. A13696-01), goat anti-human igg-HRP two anti-(purchased from sigma, article No. A8667).
Two, experimental technique
1. the structure of expression vector is selected
Select HpCHO as expression vector, build strand unit-GFP expression plasmid ANF, wherein strand unit is the single-chain antibody of anti-human T cell's surface antigen CD3, and fluorescin A is green fluorescent protein (GFP); Select pCHO as expression vector, build unit price unit-IRES – RFP expression plasmid BIF, wherein unit price unit 1 and 2 is the heavy chain light chain pairing antibody of anti-human HER2 antigen, and fluorescent protein B is red fluorescent protein (RFP).According to the multiple clone site design primer in unit price unit-IRES – RFP and strand unit-GFP gene order and pCHO1.0 and HpCHO carrier, in table 1.Wherein unit price unit with adopt Overlap extension PCR method between IRES and RFP and be connected.
Table 1PCR primer sequence
Primer I RES-F and IRXFP-R is for the IRES gene fragment that increases;
Primer HpCHOGFP-F and HpCHOGFP-R is for the GFP gene fragment that increases;
Primer I RXFP-F and HpCHOGFP-R is for the RFP gene fragment that increases;
PCR expands: the condition of IRES, GFP, RFP fragment be 95 DEG C 5 minutes, to circulate 25 times: 95 DEG C of sex change 30 seconds according to follow procedure, 56 DEG C of annealing 30 seconds, 72 DEG C extend 90 seconds, and last circulation 72 DEG C extends 10 minutes.
Overlap extension PCR method: connecting the condition of IRES and RFP is that IRES, RFP of reclaiming with purifying use primer I RES-F and HpCHOGFP-R for hybrid template, the same Standard PCR of other reagent, do not add primer, to circulate 4 times: 95 DEG C of sex change 2 minutes according to following parameters, anneal 1 minute for 55 DEG C, 72 DEG C extend 2 minutes, then add primer I RES-F and HpCHOGFP-R to circulate 25 times: 95 DEG C of sex change 30 seconds according to follow procedure, 56 DEG C of annealing are killed for 30 seconds, 72 DEG C extend 2 minutes, last circulation 72 DEG C extends 10 minutes, and the DNA fragmentation obtained is IRES-RFP.
Sepharose reclaims: after PCR primer is carried out electrophoresis, cut object band, reclaim test kit (purchased from Tiangen, article No. DP210) reclaim with sepharose, and surveys the concentration that it reclaims product.
Enzyme is cut: by reclaimed product and corresponding carrier, carry out enzyme and cut, hatch 3h for 37 DEG C with restriction enzyme.GFP and plasmid A EcoRV-HF and PacI and restriction enzyme PacI double digestion, IRES-RFP and plasmid B PacI enzyme are cut.
Connect: the fragment after being cut by enzyme and respective carrier, connect with T4DNA ligase enzyme, and condition of contact is 16 DEG C and spends the night.
2. the plasmid amplification of recombinant vectors
By the connection product conversion intestinal bacteria Trans10 of step 1, picking list bacterium colony is delivered to raw work order-checking portion, Shanghai and is checked order.Check order correct single colony inoculation in the LB substratum containing 100 μ g/ml kantlex, 37 DEG C of shaking culture 16 hours, 8000 × g collects thalline in centrifugal 10 minutes, Tiangen is used to remove the large extraction reagent kit (Tiangen of intracellular toxin, article No. DP117) extracting plasmid, after plasmid is dissolved in 100 μ l ultrapure waters, add 142 μ l Virahols and 42 μ l 5M NaCl again, plasmid is precipitated out, abandons supernatant, add 70% ethanol 0.8ml washing precipitation twice, abandon supernatant, air-dry in super clean bench, add the ultrapure water 100 μ l of sterilizing, survey its concentration and OD value.The plasmid of gained is recombinant plasmid.
3. the cotransformation of recombinant plasmid
First 24 hours of transfection, inoculates CHO-S cell, volume of culture 30ml in 125ml Erlenmeyer flask, and cell density is 0.5 × 10
6cells/ml, uses CD Forti-CHO substratum, 37 DEG C of 8%CO
2cultivate in shaking table, the cell counting on the same day of transfection, guarantees that cell viability is greater than 95%, cell density 1 × 10
6cells/ml, be then placed on by cell on shaking table and cultivate preparation transfection, before transfection, cell is not centrifugal, because centrifugal process can reduce transfection efficiency.
In transfection process, in a 1.5ml centrifuge tube, add 150 μ l OptiPRO
tMsFM substratum, then adds 5 μ l FreeStyle
tMmAX Reagent transfection reagent, adds 150 μ l OptiPRO in a 1.5ml centrifuge tube
tMsFM substratum, then adds 5 μ g plasmid DNA, soft mixing.When adding, rifle head wants below immersed in liquid level, then soft upset mixing.Join in the DNA solution of dilution by the transfection reagent solution of dilution, soft spins upside down several lower reagent bottle, but does not use vibrator, is at room temperature hatched by plasmid transfection reagent mixture and makes it form mixture in 10 minutes.Incubation time max 20 mins, by plasmid transfection reagent complex drop by drop add in the Erlenmeyer flask of culturing cell, soft the rocking of limit edged.Cell after transfection is placed on rotating speed 150rpm, 37 DEG C, the shaking table of 8%CO2 is cultivated.
4., after transfection, qualification transfection efficiency also screens with microbiotic
Transfection is after 48 hours, and flow cytometer and fluorescent microscope detect its transfection efficiency, and changes containing Totomycin (300 μ g/ml), tetracycline (5 μ g/ml), and the culture medium culturing cell of methotrexate (50nM).
5. increase with methotrexate
Be added with after vigor rises to about 85% in antibiotic substratum until cell in early stage, carry out second time pressurization, strengthen the concentration of methotrexate, carry out pressurization cultivation with the CDFortiCHO substratum of the methotrexate containing 500nM or 1000nM.
6. screen the positive cell strain of high expression level
Carry out sorting by with the express cell flow cytometer increasing out after the screening of MTX pressure, cell strong for fluorescent signal is selected, is inoculated in 96 orifice plates with the inoculum density of 1 cell per well, after cultivating two weeks, enlarged culturing.
7. flow cytometry analysis expresses the cell strain of Double antibody
Get the cell strain 200 μ l of cell viability more than 90% after enlarged culturing, dilute according to cell density PBS, be diluted to cell density and be about 0.5 × 10
6cells/ml, machine testing on flow cytometer, detects with two kinds of fluorescence channels (FL1 and FL3), analyzes, acquired results FlowJo7.6 software specifically in table 2.
Table 2 statistical study western blot analysis double antibody relative content and fluorescence intensity ratio
8. pair positive colony carries out 14 days yield assessment detection antibody productions
Cell viability after enlarged culturing is after more than 90%, and carry out yield assessment to cell strain, step is as follows:
A. 3 × 10 are inoculated
5the cell of vigor >90% (only comprises 8mM L-glutaminate and 1% resistant to aggregation reagent (Anti-Clumping Agent in 30ml fresh culture, purchased from Life technologies, article No. 0010057AE), not purine-containing mycin and MTX) in, in 37 DEG C, 8%CO
2, 150rpm shaking table is cultivated.
B. cell cultures is after 14 days, and results supernatant, centrifugal 10 minutes of 10,000rpm, gets supernatant-20 DEG C of storages after 0.22 μm of membrane filtration.
C. purify supernatant with proteinA and obtain antibody, by the output of UV spectrophotometer measuring antibody, specifically in table 3.
Table 3 ultraviolet spectrophotometer analyzes antibody production and flow cytometry analysis clone fluorescence intensity
9. detect positive colony and express double antibody relative content
The supernatant collected after cultivating 14 days, gets after 20 μ l carry out SDS-PAGE (non-reduced), carries out protein immunoblot (Western blot) and detect (Fig. 8 A).Nitrocellulose filter after transfer printing, with after ECL luminescent solution (purchased from Pierce, article No. 32106) reaction, Bio-rad chemiluminescence detector is taken pictures.Carry out band gray analysis with Imagelab software, and calculate double antibody relative content, see above-mentioned table 3; Double antibody relative content refers to that double antibody accounts for the mass percent of all antibody fragments (comprising double antibody, unit price unit, strand unit and other antibody fragments) in reconstitution cell expression product.
Analyze the dependency of clone's GFP/RFP average fluorescent strength ratio and double antibody relative content, use the Gaussian method (Fig. 8 B) of Graphpad Prism5 software.
10. detect the stability of positive colony
Positive colony is carried out continuous passage, within every two days, goes down to posterity once, passed for 30 generations continuously.In every 5 generations, carry out a flow cyctometry histogram analysis, see Fig. 9, and 14 days yield assessment, see Figure 10.Flow cyctometry histogram analysis concrete grammar is shown in step 7.Within 14 days, yield assessment method is shown in step 8.30 generation clone output are not less than 70% of 0 generation clone output and are designated as stable clone, and compare with the flow cyctometry histogram in the 0-30 generation of this clone, the results are shown in Table 4.
Table 4 different clonal cell line stability compares with flow cyctometry is histogrammic
Three, experimental result
Because the gene expression dose being connected to IRES downstream can be starkly lower than the gene expression dose being connected to IRES upstream, so all cells mono-clonal detected, the fluorescence intensity of RFP will well below the fluorescence intensity of GFP.Consider this kind of situation, for the mono-positive cell of RFP, the present embodiment is added up.Then said single positive, specially refer to that GFP is mono-positive.
As shown in Figure 5, mono-clonal there will be three kinds of situations: two positive (1#), double-negative (2#) and single positive (3#).
Fig. 6 then further illustrates the result of monoclonal cell in flow cytomery of three kinds of situations, consistent with the result of fluorescence microscope: the clone (2# of Fig. 5) that can't see any fluorescence under fluorescent microscope, flow cytometer detection is double-negative, sees Fig. 6 A, does not express fluorescence; Only see the clone (3# of Fig. 5) of green fluorescence under fluorescent microscope, flow cytometer detection is that single positive is shown in, Fig. 6 B, expresses a kind of fluorescence; Can see clone (1# of Fig. 5) that is green and red fluorescence under fluorescent microscope, flow cytometer detection is two positive, sees Fig. 6 C, expresses two kinds of fluorescence.Statistics 8 kinds of cell strain luciferase expression per-cent situations with following table 5.
8 kinds of cell strain luciferase expression per-cent situations added up by table 5
Fig. 7 shows that the antibody expression amount of cell is directly proportional to average fluorescent strength, and the cell that namely fluorescence is stronger, antibody production is also higher.
Fig. 8 shows that the double antibody relative content of cell expressing and GFP/RFP average fluorescent strength ratio meet normal distribution (R
2=0.9914), namely when GFP/RFP average fluorescent strength ratio is between 100 to 132, double antibody relative content is maximum, is more than 80%; GFP/RFP average fluorescent strength ratio is less than 100 or be greater than 132, then double antibody relative content all obviously declines.
Fig. 9 and 10 shows that the stability of positive colony cell strain can be determined by the flow cyctometry histogram of cloning in early days.FL1 passage (green fluorescence) detected result show, when flow cyctometry histogram reached for 10 generations at clonal cell line, show as unimodal form, then the expression amount in this clone 30 generation be not less than 0 generation expression amount 70%, be stable cell line.FL3 passage (red fluorescence) is because intensity is more weak, not for referencial use in the present embodiment.
Should be understood that the present invention of disclosure is not limited only to specific method, scheme and the material described, because these equal alterable.Will also be understood that terminology used here is only used to describe the object of specific embodiment scheme, instead of be intended to limit the scope of the invention, scope of the present invention is only limited to appended claim.
Those skilled in the art also will recognize, or can confirm that use is no more than normal experiment, many Equivalents of specific embodiment of the present invention described in this article.These Equivalents are intended to comprise in the appended claims.
Claims (13)
1. the method for bi-specific antibody cell strain is expressed in preparation and screening, it is characterized in that comprising the following steps:
Step 1: two plasmids of construction expression bi-specific antibody, described two plasmids all contain double-promoter, and the albumen of one of them plasmid expression is one or two polypeptide of specific combination tumour antigen; The albumen of another plasmid expression is one or two polypeptide of specific combination immunologically competent cell antigen; Described two plasmids containing double-promoter also express a kind of different fluorescin respectively; If the albumen of plasmid expression is two polypeptide forms, then fluorescin utilizes internal ribosomal entry site sequence to be connected to the C end of a polypeptide, if a polypeptide form, then fluorescin is directly inserted into another promotor multicloning sites downstream of carrier;
Step 2: obtain in step 1 two kinds of recombinant plasmids are carried out transforming, cultivate and extracting;
Step 3: by two kinds of recombinant plasmid cotransfections of extraction in step 2 in host cell;
Step 4: the positive monoclonal cell strain of two fluorescence is expressed in screening.
2. described method according to claim 1, is characterized in that: if the albumen of described plasmid expression is a polypeptide, then on plasmid, a promotor is used for initiation transcription protein unit, and another promotor is used for initiation transcription fluorescin; If the albumen of plasmid expression is two polypeptide, then 3 ' of the DNA encoding sequence of a polypeptide on plasmid to connect the DNA encoding sequence of fluorescin by IRES.
3. described method according to claim 1, is characterized in that: two kinds of recombinant plasmids transformation of E. coli respectively in described step 2.
4. described method according to claim 1, is characterized in that: in described step 2, also comprise the plasmid that axenic purification extracts.
5. described method according to claim 1, is characterized in that: in described step 3, also comprise interpolation screening pressure; Preferably described screening pressure is tetracycline, Totomycin or methotrexate.
6. described method according to claim 1, is characterized in that: screen with flow cytometer or fluorescent microscope in described step 4.
7. described method according to claim 1, is characterized in that: also comprise the positive monoclonal cell strain after by screening and carry out enlarged culturing, detect its fluorescence intensity with flow cytometer or fluorescent microscope.
8. described method according to claim 1, is characterized in that: also comprise the positive monoclonal cell strain after to screening and utilize flow cytometry to carry out Rapid Stability assessment, determine the stability of monoclonal cell strain within 10 generations.
9. described method according to claim 1, is characterized in that: described host cell is mammalian cell; Preferably, described mammalian cell is Chinese hamster ovary cell.
10. described method according to claim 1, is characterized in that: described fluorescin correctly can indicate protein expression situation after transfection, is green fluorescent protein, red fluorescent protein, yellow fluorescence protein, orange fluorescent protein or blue fluorescent protein.
11. described methods according to claim 1, is characterized in that: in the expression product of described bi-specific antibody cell strain, double antibody relative content is assessed by the average fluorescent strength ratio of calculating two kinds of fluorescence.
12. described methods according to claim 1, is characterized in that: the stability of described double antibody cell strain expression level is assessed by the distribution of flow cyctometry analysis of cells fluorescence intensity.
The 13. expression bi-specific antibody cell strains prepared according to the arbitrary described method of claim 1-12 and screen.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN115074332A (en) * | 2022-06-21 | 2022-09-20 | 中国人民解放军陆军军医大学第一附属医院 | Method for constructing Huh7-eGFP cell strain for stably expressing green fluorescent protein |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111394387A (en) * | 2020-03-13 | 2020-07-10 | 苏州智享众创孵化管理有限公司 | Construction and screening method of bispecific antibody cell strain |
| WO2022095311A1 (en) * | 2020-11-03 | 2022-05-12 | 中国科学院苏州纳米技术与纳米仿生研究所 | Screening method |
| US11693012B1 (en) | 2020-11-03 | 2023-07-04 | Suzhou Institute Of Nano-Tech And Nano-Bionics (Sinano) , Chinese Academy Of Sciences | Screening method |
| CN113337452A (en) * | 2021-04-19 | 2021-09-03 | 中国医学科学院医学生物学研究所 | Method for preparing double-factor antibody by serum-free culture expression of high-density CHO-S cells |
| EP4239333A1 (en) * | 2022-03-03 | 2023-09-06 | Lonza Biologics plc | Method for selecting clonal cells expressing correctly assembled multi-specific binding molecules |
| WO2023166130A1 (en) * | 2022-03-03 | 2023-09-07 | Lonza Biologics Plc. | Method for selecting clonal cells expressing correctly assembled multi-specific binding molecules |
| CN115074332A (en) * | 2022-06-21 | 2022-09-20 | 中国人民解放军陆军军医大学第一附属医院 | Method for constructing Huh7-eGFP cell strain for stably expressing green fluorescent protein |
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