CN106699886A - Antibody dependent cell mediated cytotoxicity (ADCC) enhanced ofatumumab antibody - Google Patents
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Abstract
Provided is an antibody dependent cell mediated cytotoxicity (ADCC) enhanced ofatumumab antibody. The invention discloses the antibody having ADCC (antibody dependent cell mediated cytotoxicity) activity significantly improved by changing the composition of a sugar chain of ofatumumab; the significantly improved activity of the antibody is confirmed in an animal body and ofatumumab head-to-head studies. Being different from a Biowa company Potelligent technology and a Roche GlycoMab technology performing transformation at a cellular level through genetic engineering, the antibody has the means of adding fucose analogues in a culture process in a ground-breaking manner, the metabolic pathway of the cells is effectively regulated, the fucose content in the target monoclonal antibody can be reduced or fucose can be removed, and the purpose of significantly improving the monoclonal antibody ADCC activity is achieved.
Description
Technical field
The invention belongs to genetic engineering field, be related to one pass through to change listed method difficult to understand wood (Ofatumumab,
Arzerra) the sugar chain composition of monoclonal antibody, obtains the enhanced antibody of cytotoxicity conspicuousness of antibody dependent cellular mediation.This
Invention also covers the preparation method and its chronic lymphatic knurl in treatment tumour, especially CD20 expression high of the antibody,
Application in non Hodgkin lymphom and diffusivity large B cell lymphoid tumor.
Background technology
CD20 (the also known as restricted differentiation antigen of human B lymphocyte) is on the bone-marrow-derived lymphocyte of pre B cell Nuclear maturity
Hydrophobic transmembrane protein (Valentine et al., J.Bio.Chem., 1989, V264:11282-7).Research finds, exceedes
90% non Hodgkin lymphom patient, chronic lymphatic knurl patient and large B cell lymphoid tumor patients B cells' apparent height
Expression CD20 molecules (Anderson et al., Blood, 1984, V63:1424-33), but candidate stem cell, pro B lymphocyte,
Normal plasma cells and other tissue in do not find preceding feature (Tedder et al., Immunology, 1985,
V135:973-9).Based on this feature, Biogen companies had listed that specificity is embedding for the people mouse of CD20 in 1997
Chimeric antibody Rituxan (Rituximab), for treating chronic lymphatic knurl, non Hodgkin lymphom etc., and in clinic
The upper goldstandard for turning into treatment.Genmab companies then had listed the full people for CD20 size ring regions in 2009
Antibody-difficult to understand (ofatumumab), the CDC (CDC) of the antibody is far above Rituxan,
The first-line treatment for chronic B cell lymthoma is ratified by FDA, the current kind carries out non-Hodgkins pouring
The clinical research of bar knurl first-line treatment.
The enhanced third generation IDEC-C2B8 Gavyza of cytotoxicity (ADCC) of current antibody dependent cellular mediation
(obinutuzumab) inundatory clinical advantage is shown in Rituxan clinical tests head to head, it was demonstrated that
ADCC activity occupies the status of unusual core in the Antybody therapy with CD20 as target spot.Herter etc. is to method difficult to understand wood
The head to head comparative study that monoclonal antibody (ofatumumab) is carried out with Rituxan shows, difficult to understand is in vitro and animal
Internal every activity index is no less than Rituxan, and its CDC (CDC) is far above Rituxan
(Herter et al.,Mol.Cancer.Ther.2013；12:2031-42).Therefore, the anti-of difficult to understand is further improved
The cytotoxicity of body dependent cell mediation, will assign its prior clinical meaning.
The content of the invention
Lived in ADCC based on outstanding role of the ADCC activity in the treatment of CD20 antibody clinicals, and difficult to understand
Property room for promotion and possibility, the present invention by change the sugar chain of method wood (ofatumumab) monoclonal antibody difficult to understand constitute lifting its
BA, obtains a new antibodies with BA higher.The lifting of its antitumor activity is in the present invention
The internal test of pesticide effectiveness of the in vitro test and animal level that have passed through cellular level is confirmed.
Likewise it is preferred that CHO expression systems substitute NS0 expression systems, so as to reduce or eliminate NS0 system expressions Austria method
The Di- α 1 contained during wooden monoclonal antibody, 3GalGal sugar-modified component makes monoclonal antibody of the present invention have lower immunogenicity；
Application it is a further object to provide the preparation method of above-mentioned monoclonal antibody and its in antineoplastic.
The present invention is realized by following technical measures：
One by change the sugar chain of method wood (ofatumumab) monoclonal antibody difficult to understand constitute lifted its BA antibody and its
Preparation method.
According to the antibody, it is characterised in that：Its primary amino acid sequences and method wood (ofatumumab) monoclonal antibody one-level difficult to understand
Consensus amino acid sequence, but the sugar chain of its modified antibodies during expression is different from modification difficult to understand sugar chain, makes it
BA method wood antibody more difficult to understand is significantly increased.
According to the antibody, it is characterised in that：By fucose content in the sugar chain for reducing modified antibodies, enhancing antibody according to
The cytotoxicity for relying property cell-mediated.
According to the antibody, it is characterised in that：In the sugar chain of the modified antibodies being made up of carbohydrate, has fucose
The mass percent of sugar chain total sugar chain for accounting for modified antibodies be not higher than 20%.
According to the antibody, further characterized in that：In the sugar chain of the modified antibodies being made up of carbohydrate, tool
The mass percent that the sugar chain of fucose accounts for total sugar chain of modified antibodies is not higher than 5%.
According to the antibody, further characterized in that：In the sugar chain of the modified antibodies being made up of carbohydrate, no
Containing fucose.
According to the antibody, it is characterised in that turned in the culture medium that with the addition of L-fucose analog by eukaryotic host cell
Translate expression.
According to the fucose analogue, preferably 5- alkynyls-L-fucose, the fluoro- L-fucoses of 2- deoxidations -2- and their phases
That answers crosses acylated derivatives.
According to the fucose analogue, the preferably fluoro- L-fucoses of 2- deoxidations -2- and corresponding acylated derivatives excessively.
According to the fucose analogue, preferably 2F-Peracetyl-Fucose.
According to the host cell, preferably NS0 cell lines, SP2/0 cell lines and Chinese hamster ovary celI system.
According to the host cell, preferably Chinese hamster ovary celI system.
A kind of composition, the composition includes above-mentioned antibody.
Described antibody and preparation method thereof is preparing the application of antineoplastic.
Described antineoplastic and its application, preferably chronic lymphatic knurl, non Hodgkin lymphom and the big B of diffusivity are thin
Born of the same parents' lymthoma.
Key step of the invention is below mainly provided：
The major technique that purpose monoclonal antibody ADCC activity is lifted by reducing or removing fucose is to concentrate on cytogene water
Flat bed is transformed on face, such as POTELLIGENT (R) technologies and the GlycoMAb technologies of Roche of Biowa companies.
Breakthrough being realized by adjusting the metabolic pathway of cell of the present invention reduces or removes fucose content in purpose monoclonal antibody,
As added fucose analogue in incubation, the sugar chain of modified antibodies is made up of technology controlling and process is adjusted, reached
Conspicuousness lifts the purpose of monoclonal antibody ADCC activity.
Antibody is needed with GDP-fucose as substrate in mammalian cell, and α -1,6 are carried out by fucosyl transferase
Fucose is modified, and from the beginning intracellular GDP-fucose (can synthesize) approach or using free by using glucose
Fucose from remedial pathway generate.From the beginning the GDP-fucose levels of intracellular can be to (synthesizing) enzyme (such as GMD) of approach
Generation feedback inhibition, or suppress the activity of fucosyl transferase, so as to influence core fucose to modify.By in culture
During add fucose analogue, such as 2F-Peracetyl-Fucose (abbreviation 2F), form GDP-2F-fucose, suppression
The synthesis of normal GDP-fucose is made, and suppresses fucosyl transferase activity, so as to obtain low fucose content
Sugar chain antibody, the antibody has ADCC activity higher compared with difficult to understand.Meanwhile, the method is relatively by genetically engineered
Engineered cells (such as Glycart Biotechnology AG applies for a patent method, CN02818173.5) are more convenient, resist
Body yield is also dramatically increased.
Beneficial effects of the present invention
The main advantage of the present invention：The antibody of acquisition keeps the security similar to difficult to understand simultaneously, and acquisition is higher
ADCC activity, this change is clinically by beneficial reduction because there is the probability that therapeutic is resisted in patient's antagonist.Base
In the monoclonal antibody prepared by the present invention, existing difficult to understand is all significantly higher than in terms of clinical drug effect and security, had
There is significant advance, also comply with introduction, the utility strategies of digestion re-absorption in terms of China's biological medicine exploitation, reduce
The risk of drug development.
Brief description of the drawings
The antibody A DCC of Figure 12 F-Peracetyl-Fucose treatment lifts the MS collection of illustrative plates of monoclonal antibody.
The MS collection of illustrative plates of the antibody of Figure 25-Alkynyl-Fucose treatment
The difficult to understand MS collection of illustrative plates of Fig. 3 CHO expression.
Fig. 4 originals grind difficult to understand MS collection of illustrative plates.
The schematic diagram of Fig. 5 antibody modification sugar chains.
Fig. 6 difficult to understand is compared by modifying tumor killing effect in front and rear its animal body of sugar chain ADCC activity change
Specific embodiment
With reference to specific embodiment, the present invention is further elaborated, but does not limit the present invention.Here, we demonstrating
The antibody prepared in the present invention is compared with difficult to understand：By reducing the fucose content of modification sugar chain in antibody, obtain
Compared with the antibody that difficult to understand has ADCC activity higher, stronger antitumor activity is illustrated in vivo.
The different fucose analogues of embodiment 1 prepare the antibody of fucose knockout
In the present invention, cell line ET-4-66 (its in CHO (DG44) sources based on stabilization expression difficult to understand
Screening process refers to roc exhibition etc., to Mongolian and the human antibody of the anti-CD20 of Caucasoid's low immunogenicity, application
Number CN201410003725.6, entire contents are literary incorporated by reference herein), by regulating and controlling generation of the fucose in its cell
Apologize for having done sth. wrong journey, obtain the sugar chain antibody of low fucose content.
Recovery：The cryopreservation tube one (loading amount 1.5ml) for having ET-4-66 working cardial cells, 37 DEG C of water are taken from liquid nitrogen container
Bath is thawed, and seed liquor is transferred in the 125mL shaking flasks containing 20-30mL CD FortiCHO culture mediums immediately.To shake
Bottle is positioned in CO2 incubators, 37 DEG C, 8%CO2, 130rpm cultures.
Inoculation：Take the logarithm growth period cell according to 0.8-1.2 × 106Cells/mL cell densities are inoculated in 125mL shaking flasks
In, volume of culture 30mL adds 200 μM, 100 μM, 50 μM of 2F-Peracetyl-Fucose respectively in the medium
And 5-Alkynyl-L-fucose, culture to 4-5 × 106During cells/mL density, by 0.8-1.2 × 106Cells/mL is again
Continuous culture 9 days is inoculated in the fresh culture containing above-mentioned fucose analogue, and culture supernatant is obtained through affinitive layer purification
To the antibody that algae sugar is knocked out, with the comparative studies that difficult to understand carries out sugar and activity.
The ADCC activity Antibody preparation high of embodiment 2
Recovery：The cryopreservation tube one (loading amount 1.5ml) for having ET-4-66 working cardial cells, 37 DEG C of water are taken from liquid nitrogen container
Bath is thawed, and seed liquor is transferred into the 125mL containing 20-30mL FortiCHO (addition MTX500nM) culture medium immediately
In shaking flask.Shaking flask is positioned in CO2 incubators, 37 DEG C, 8%CO2, 130rpm cultures 2-3 days.
Shaking flask expanding species：When cell density increases to 1.5-3.5 × 106During cells/mL, expanding species is carried out to cell.During operation
As needed, appropriate fresh FortiCHO culture mediums are supplemented, the density of cell is in 0.4-0.6 × 10 after expanding species6
cells/mL.Shaking flask, 1/3 of volume of culture no more than shaking flask cumulative volume are changed according to cell liquid volume..In N-1 generations, start
2F-Peracetyl-Fucose cultures (N refers to the cell culture generation of production) of 50 μM of addition, for reactor experiment.
With 0.8-1.2 × 106Cells/mL cell densities be inoculated with, according to (be shown in Table 1) in experimental program design feeding strategy
It is added with condition of culture and is controlled, reactor parameter control is as follows：
Working volume：Initial 1.2L culture mediums
Temperature：Control is at 36.5 DEG C
pH：Setting 7.0 ± 0.05, uses CO respectively2With 0.5M NaHCO3PID/feedback is controlled
DO：40% (oxygen is 100% with air) is set, is controlled with oxygen PID/feedback
Rotating speed：Control 80-250rpm, initial rotating speed 80rpm, 1hr after inoculation, rotating speed is improved to 150rpm, the
Rotating speed improves 200rpm within 5 days, if sparger throughputs are more than 100ml/min, rotating speed can be improved to 250rpm.
Gas is controlled：Overlay compressed airs 40-150ml/min
Sparger CO2PID/feedback control pH setting upper limits 300ml/min
O2PID/feedback control DO setting upper limits 300ml/min
Sampling daily, observation of cell growth conditions calculate living cells quantity and Cell viability, and biochemical point is carried out with Nova
Analysis, and pipette and be centrifuged under the conditions of 1000rpm, 3min more than 1ml samples, take -80 DEG C of supernatant and preserve.
When Cell viability is less than 70%, or viable count is when being the half of peak value, stops culture harvesting, centrifugation
Leave and take supernatant purifying.Purifying key step include in-depth filtration, affinity chromatography, acid be incubated inactivation, cation-exchange chromatography,
Anion-exchange chromatography, nanofiltration and ultrafiltration step.ADCC lifting antibody after purification carries out effect experiment in animal body
The sugar-type analysis of the antibody of the present invention of embodiment 3
The parsing of sugar-type is based primarily upon ESI-QTOF antagonists heavy chain and is analyzed with the sugar chain that combining form is present, Fig. 1
It is that the monoclonal antibody and original knocked out to fucose respectively based on QSTAR XL (AB Sciex) mass spectrograph grind product Austria method wood to Fig. 3
The test sugar spectrum of monoclonal antibody.It is anti-that the antibody (Fig. 1) of 2F-Peracetyl-Fucose treatment, 5-Alkynyl-L-fucose are processed
It is specific that body (Fig. 2), difficult to understand (Fig. 3) and the original of CHO expression grind difficult to understand (Fig. 4) its MS collection of illustrative plates
Parsing is shown in Table 2.
Knowable to the MASS results of table 2, product and untreated antibody are ground compared to method difficult to understand wood original, by adding in incubation
Plus 2F-Peracetyl-Fucose and 5-Alkynyl-L-fucose change the modification sugar chain of antibody completely.In mass spectrogram upper table
Now for the molecular weight at each main signal peak is the molecular weight (2 that 292Da has all been subtracted on the basis of the main peak type that original grinds product
Individual fucosyl residues molecular weight).By confirmation, it is determined that all having taken off 2 fucoses (because monoclonal antibody is 2 identical heavy
Chain is linked together by interchain disulfide bond), so in incubation, addition 2F-Peracetyl-Fucose and
5-Alkynyl-L-fucose can effectively remove fucose.
The comparative analysis of the antibody A DCC activity of the present invention of embodiment 4
ADCC effects are determined to be carried out based on engineered NK cells, determines killing of the antibody to Wil2-s cells.
Wil2-s target cells prepare：Target cell Wil2-s, 1000rpm in exponential phase are taken, are centrifuged 5 minutes,
Be washed once with RPMI 1640 (#11835), adjustment cell concentration is 5 × 105/ ml, (removes in 96 hole round bottom plates
Outside effector cell's autofluorescence control wells and blank background control wells) 50 μ l are added per hole；
Original ground into medicine with RPMI1640 or other detected samples are diluted to 1 μ g/ml, then do 5 times of gradient dilutions, it is dilute
9 points are released, a blank spot without antibody is increased in addition；96 orifice plates add 50 μ l to mix with target cell per hole, often
Individual concentration does 2 multiple holes；
At 37 DEG C, 5%CO2 is incubated 30 minutes 96 orifice plates；
NK-92MI/CD16a effector cell：Take the logarithm effector cell NK-92MI/CD16a, the 1000rpm in growth period,
Centrifugation 5 minutes, washed once with RPMI 1640, and adjustment cell concentration is 5 × 105/ ml, (removes in 96 hole round bottom plates
Outside target cell autofluorescence control wells and the maximum cracking control wells of target cell) 100 μ l are added per hole；
At 37 DEG C, 5%CO2 is incubated 4 hours 96 orifice plates；
Fluorescence signal is detected with CytoTox-ONE kits：
A) 96 orifice plates place room temperature after being incubated and terminating, and balance 20~30 minutes；All detection reagents are first balanced to room
Temperature；
B) 4 μ l lysates are added per hole in the maximum cracking hole of target cell；
C) 96 orifice plate 1000rpm, is centrifuged 5 minutes；
D) the μ l of supernatant 100 are taken, 96 hole blackboards (#3904) are moved to；
E) 100 μ l reaction substrates are added per hole, room temperature is placed 10 minutes；
F) 50 μ l reaction terminating liquids are added per hole, is well mixed.
Fluorescence Ex560nm/Em590nm signals are recorded in ELIASA SpectraMax M5.
Data processing：
A) all values should first subtract blank class value；[(experimental group-target is thin to calculate killing rate ADCC%=by formula below
The spontaneous spontaneous release group of release group-effector cell of born of the same parents)/(the target cell maximum release group-spontaneous release of target cell
Group)] × 100%
B) X-axis data grind the mass concentration (ng/ml) of medicine for the original of log conversions, and Y-axis data are ADCC%.
C) make nonlinear regression with GraphPad Prism 5, use 4 parameter equations f (x)=B+
(A-B)/(1+10^ ((C-x) * D)) fitting obtains the regression curve that test sample kills Wil2-s cells.Calculate
EC50, R2 etc..
From table 3 it can be seen that by adding fucose substitute 2F-Peracetyl-Fucose and 5-Alkynyl-L-fucose,
Change the metabolic fluxes of cell line, so as to prepare the antibody of ADCC activity high, the ADCC activity (EC of the antibody50
It is 0.1718~0.3372ng/mL) similar (EC of the Gazyva monoclonal antibodies of ADCC newly approved to Roche lifting50It is 0.3836
Ng/mL), grind difficult to understand far above original and CHO express difficult to understand ADCC activity (EC50Respectively
It is 2.718ng/mL and 3.097ng/mL).So as to support described in this patent：Adjusted by adding fucose analogue
The metabolism of cell is controlled, so as to obtain the antibody of low fucose content.
The evaluation of the antibody activity in vivo of the present invention of embodiment 5
BALB/cA-nude nude mouses, 6-7 weeks, ♀, nude mouse subcutaneous vaccination human B cell lymthoma Daudi cells,
Treat tumour growth to 100-150mm3Afterwards, animal is grouped (D0) at random.Dosage and dosage regimen are shown in Table 4.Weekly
2-3 knurl volume is surveyed, claims mouse weight, record data.Gross tumor volume (V) computing formula is：
V=1/2 × a × b2Wherein a, b represent length and width respectively.
T/C (%)=(T-T0)/(C-C0) × 100% wherein T, C are the gross tumor volume at the end of experiment；T0、C0For experiment starts
When gross tumor volume.
Dosage regimen and experimental result are shown in Table 4 and Fig. 6.Difficult to understand, ADCC lifting monoclonal antibody (3mg/kg, IV, often
Week 2 times, totally 4 times) the obvious growth for suppressing CD20 positive B-cells lymthoma Daudi nude mouse subcutaneous transplantation knurls,
Tumour inhibiting rate is respectively 84% and 95%, and difficult to understand group has 2/6 tumor partial regression；ADCC liftings antibodyome has
2/6 tumor partial regression and 1/6 completed tumor regression；The suppression of control drug Gazyva same doses and scheme to Daudi
Ratio of outflow is 89%, there is 1/6 tumor partial regression and 1/6 completed tumor regression；Another reference agent Mabthera(30mg/kg,
IV, 2 times a week, totally 4 times) to the tumour inhibiting rate of Daudi it is 109%, there are 1/6 tumor partial regression and 2/6 tumour complete
Disappear.Tumor-bearing mice can be tolerated very well to above medicine, not have the symptoms such as weight loss.ADCC lifts antibody
Curative effect to Daudi is better than method wood difficult to understand, similar to Gazyva.
The 2F of table 1 is applied to the design of 3L reactor performances test experiments
Remarks：Glucose control ＞ 2.5g/L；
EFC：Beginning in 3rd day adds 3% (V/V) online by initialization volume daily, adds total amount for 33-36%；
Tyr：7th day addition 1/500 (V/V), the 13rd day 1/1000 (V/V) of addition；
Cys：7th day addition 1/100 (V/V), the 13rd day 1/200 (V/V) of addition；
The difficult to understand of table 2 is parsed with the MS signal peaks of ADCC lifting samples
The ADCC results that the cellular level of table 3. is determined
The curative effect of each antibody on human B cell lymphoma Daudi nude mouses subcutaneous transplantation knurl of table 4..
Note：D0:First time administration time；P values refer to compared with solvent；Checked using Student ' s t.Mouse number when experiment starts：Solvent
Group n=12, treatment group n=6.
Claims (15)
1. one passes through to change method wood difficult to understand（ofatumumab）The sugar chain composition of monoclonal antibody lifts the antibody of its BA.
2. antibody according to claim 1, it is characterised in that：Its primary amino acid sequences and method wood difficult to understand（ofatumumab）Monoclonal antibody primary amino acid sequences are consistent, but the sugar chain of its modified antibodies during expression is different from the sugar chain of modification difficult to understand, significantly increase its BA method wood antibody more difficult to understand.
3. antibody according to claim 2, it is characterised in that：By fucose content in the sugar chain for reducing modified antibodies, strengthen the cytotoxicity of antibody dependent cellular mediation.
4. antibody according to claim 3, it is characterised in that：In the sugar chain of the modified antibodies being made up of carbohydrate, the mass percent of total sugar chain that the sugar chain for having fucose accounts for modified antibodies is not higher than 20%.
5. antibody according to claim 4, further characterized in that：In the sugar chain of the modified antibodies being made up of carbohydrate, the mass percent of total sugar chain that the sugar chain for having fucose accounts for modified antibodies is not higher than 5%.
6. antibody according to claim 5, further characterized in that：In the sugar chain of the modified antibodies being made up of carbohydrate, without fucose.
7. the preparation of the antibody according to claim 1-6, it is characterised in that by eukaryotic host cell in the culture medium that with the addition of L-fucose analog accurate translation.
8. fucose analogue according to claim 7, preferably 5- alkynyls-L-fucose, the fluoro- L-fucoses of 2- deoxidations -2- and they are corresponding crosses acylated derivatives.
9. fucose analogue according to claim 8, preferably the fluoro- L-fucoses of 2- deoxidations -2- and corresponding crosses acylated derivatives.
10. fucose analogue according to claim 9, the preferably fluoro- L-fucoses of 2- deoxidations -2-.
11. host cells according to claim 7, preferably NS0 cell lines, SP2/0 cell lines and Chinese hamster ovary celI system.
12. host cells according to claim 11, preferably Chinese hamster ovary celI system.
A kind of 13. compositions, the composition includes antibody described in claim 1-6.
Antibody and preparation method in 14. claim 1-13 described in any one are preparing the application of antineoplastic.
Antineoplastic and its application described in 15. claim 13-14, preferably chronic lymphatic knurl, non Hodgkin lymphom and diffusivity large B cell lymphoid tumor.
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|---|---|---|---|
| CN201510406202.0A CN106699886A (en) | 2015-07-13 | 2015-07-13 | Antibody dependent cell mediated cytotoxicity (ADCC) enhanced ofatumumab antibody |
| PCT/CN2016/089820 WO2017008736A1 (en) | 2015-07-13 | 2016-07-12 | Ofatumumab having enhanced antibody-dependent cell-mediated cytotoxicity |
| CN201680028044.2A CN108473570A (en) | 2015-07-13 | 2016-07-12 | The method wood antibody difficult to understand of the cytotoxicity enhancing of antibody dependent cellular mediation |
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| CN201510406202.0A CN106699886A (en) | 2015-07-13 | 2015-07-13 | Antibody dependent cell mediated cytotoxicity (ADCC) enhanced ofatumumab antibody |
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|---|---|
| CN108473570A (en) | 2018-08-31 |
| WO2017008736A1 (en) | 2017-01-19 |
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