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Process Biochemistry 46 (2011) 858–863

Contents lists available at ScienceDirect

Process Biochemistry

journa l homepage: www.e lsev ier .com/ locate /procbio

ransient gene expression of a mouse homolog of Fc�-Fc� fusion protein fornti-allergic function assay

u Chenga, Xiangming Sunb, Xiaoping Yia, Yuanxing Zhanga,∗

State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, ChinaShanghai Fuchun Kexin Biotech Co. Ltd., Shanghai 201203, China

r t i c l e i n f o

rticle history:eceived 20 October 2010eceived in revised form 5 December 2010ccepted 12 December 2010

eywords:nti-allergy fusion protein (AAFP)

a b s t r a c t

The Fc�∼Fc� fusion protein (AAFP) is considered as a promising drug candidate for IgE-mediated allergicdiseases by bridging Fc�RI and Fc�RII together on mast cells and basophils. The coaggregation depends onthe flexible IgG1 hinge in AAFP with a necessary spatial relationship between AAFP and its two receptors.In this work, a mouse homolog of AAFP (mAAFP) was designed and transiently expressed in Chinesehamster ovary cells to facilitate the evaluation of its efficacy in mouse. The parameters affecting transientgene expression (TGE) were optimized in 6-well plates, and then the process was conducted to produce

ransient gene expressionHO cellsCA

2 mg mAAFP in the batch culture in 1.3 l bioreactor. To improve the production yield, fed-batch andhypothermic fed-batch cultures were adopted to achieve 5.5- and 14-fold increases, respectively. Theobtained mAAFP was assayed in vivo in a mouse passive cutaneous anaphylaxis (PCA) model. The resultsshowed that mAAFP significantly inhibited IgE-mediated allergic reaction and its efficacy lasted for atleast 24 days. It is indicated that the IgG1 hinge could coaggregate Fc�RI with the inhibitory receptorFc�RII on mast cells and basophils, and thus activate the inhibitory signaling pathway of Fc�RII. This

at po

work shows AAFP has gre

. Introduction

The fusion protein GE2, which is sequentially comprised of IgG1c, synthetic 15-amino acid linker and IgE Fc, has demonstrated itsreat potential for therapy of IgE-mediated allergy [1]. This therapyepends on the ability of GE2 to initiate the inhibitory signaling byc�RII after coaggregation of Fc�RI with Fc�RII on mast cells andasophils. The efficacy of GE2 in asthma treatment has been furthererified in monkeys [2,3]. However, GE2 efficacy may be under-ined when used in human due to the potential immunity of the

on-human linker between Fc� and Fc�. Another Fc�∼Fc� fusionrotein (AAFP) without any non-human linker was constructed by

inking Fc� (CH2–CH3–CH4) directly with Fc� (Hinge–CH2–CH3) [4].he flexible IgG1 hinge in AAFP may act as the linker in GE2 enablingAFP to simultaneously bind with Fc�RI and Fc�RII on the cells. But,

t is concerned whether or not AAFP will be as efficacious as GE2hen using IgG1 hinge instead of the synthetic linker. Furthermore,

t is difficult to conduct detailed mechanistic and efficacy studiesf AAFP on human cells for therapy of allergic diseases, whereashese studies can be facilitated by use of a mouse homology of AAFPmAAFP) in mouse.

∗ Corresponding author. Tel.: +86 21 64253065; fax: +86 21 64253025.E-mail address: yxzhang@ecust.edu.cn (Y. Zhang).

359-5113/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.oi:10.1016/j.procbio.2010.12.006

tential for allergy therapy.© 2010 Elsevier Ltd. All rights reserved.

Transient gene expression (TGE) has been frequently used forthe fast production of milligram to gram quantities of recombi-nant proteins in mammalian cells [5,6]. As reported, the proteintiter of TGE in various cell lines could reach 10–80 mg/l [7–9], andthe process has been scaled up to more than 100 l [10–12]. How-ever, it is very difficult to use an established process for producinga new protein with the same productivity because the process isprotein-dependent. The TGE process is rather complicated and itsefficiency is affected by many factors, including host cells, DNAcarrier, serum-free medium and transfection process. HEK 293cells were extensively used for transient transfection due to thegood transfectability [13–15]. However, CHO cells have becomemore favorable at present because a large number of recombinantproteins produced for clinical trials were expressed in stably trans-fected CHO cells [16–19]. To avoid the variation in the recombinantprotein derived from different host species, we have to expendmuch effort to optimize CHO-based TGE.

In this work, combination use of CDAGT-CHO and PF-CHO mediawas applied in TGE for the first time and then an optimal TGEprocess was established for mAAFP production in CHO-S cells.

This process was conducted in 1.3 l bioreactor and up to 14-foldincrease in yield was achieved by hypothermic fed-batch culture.The obtained mAAFP was then tested in a mouse passive cuta-neous anaphylaxis (PCA) model to show its therapeutic potentialin IgE-mediated diseases.

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. Materials and methods

.1. Cell line and culture

CHO-S (Invitrogen, Carlsbad, CA, USA) cells were grown in CDAGT-CHO mediumGibco, Carlsbad, CA, USA) and passaged every 48 h in shake flasks at a seedingensity of 1.0 × 106 cells/ml. The flasks were placed on the shaker platform rotatingt 125 rpm in an incubator (37 ◦C, 5% CO2).

.2. Plasmids

The mAAFP DNA, composed of mouse IgE Fc (GenBank ID: J00476.1) linked toouse IgG1 Fc (GenBank ID: L27437.1), was first synthesized (Generay Biotech. Co.,

hanghai, China), and then cloned into the plasmid pIRES (Clontech, Palo Alto, CA,SA) between EcoR I and Sal I. The resultant plasmid was designated pI-EG and used

or mAAFP production. The plasmid pCEP4-EGFP was constructed by inserting EGFPene into the vector pCEP4 (Invitrogen) between Hind III and BamH I to express EGFPs a reporter. The plasmid DNA was prepared by the alkaline lysis method [20]. DNAoncentration was quantified by a spectrophotometer (Nanodrop ND1000, Thermoisher Scientific, Wilmington, DE, USA). Only the plasmid DNA with an A260/A280

atio between 1.80 and 1.95 was used for transfection.

.3. Transfection agents

Stock solution of linear 25 kDa PEI (Polysciences, Warrington, PA, USA) andranched 25 kDa PEI (Sigma–Aldrich, St Louis, MO, USA) were prepared in ultra-pureater at a final concentration of 2 mg/ml, respectively. The solution was neutralized

o pH 7.0 with HCl before sterilization by passing through a 0.22 �m filter and storedt 4 ◦C.

.4. Transient transfection in 6-well plates

The transfection optimization was carried out in 6-well plates with pCEP4-GFP and pI-EG in parallel. The cells were counted and resuspended in CDAGT-CHOedium at 1.0 × 106 cells/ml and plated in 6-well plates (BD FalconTM, Franklin

akes, NJ, USA) with 1 ml per well. A required amount of plasmid DNA and the corre-ponding amount of PEI were added into a certain volume of medium and mixed byortexing. The DNA/PEI mixture was incubated for some minutes before added tohe culture. The culture was then incubated at 37 ◦C on a shaker platform rotating at25 rpm in a 5% CO2 atmosphere. At 6 h post-transfection, 1 ml of PF-CHO mediumHyclone, Thermo Science, USA) was topped up to the culture.

.5. Transient transfection in bioreactor

To produce mAAFP, the transient transfection was conducted in a 1.3 l bioreactorBioFlo 110, New Brunswick Scientific Co. Inc., Edison, NJ). CHO-S cells were seededt a density of 2.0 × 106 cells/ml with an initial volume of 450 ml. DNA/PEI complexas prepared and added to the culture as described above. At 6 h post-transfection,

50 ml PF-CHO medium was topped up. When the residual glucose reduced to <1 g/l,eeding was started to maintain the glucose concentration at 1–2 g/l. In some cases,he culture temperature was lowered to 32 ◦C when cell density reached around× 106 cells/ml.

.6. Analysis

Transfection efficiency, defined as the percentage of GFP-positive cells at 48 host-transfection, was measured on a BD FACSCalibur cytometer (Becton Dickin-on Immunocytometry Systems, San Jose, CA, USA). Briefly, cells transfected withCEP4-EGFP were collected by centrifugation at 1000 rpm for 5 min and washedith phosphate buffer saline solution (PBS, pH 7.0), then resuspended in cold PBS

efore assay on the cytometer.The expression level of mAAFP was measured by a sandwich ELISA using a

oat anti-mouse IgG antibody (Cellway-Lab, Luoyang, China) for capture and aeroxidase-conjugated goat anti-mouse IgG Fc (Sigma) for color development. Cellensity and viability were determined using the trypan blue exclusion method onhaemocytometer.

.7. Passive cutaneous anaphylaxis

Kun-ming mice were first injected subcutaneously with mAAFP at dosage ofmg/kg, 0.68 mg/kg, 2.0 mg/kg and 6.13 mg/kg on the back neck separately. Twenty

our hours later, mouse IgE anti-dinitrophenyl (110 ng, Sigma–Aldrich) was injectedntradermally into the right ear, and physiological saline into the left as control. Afterh, the mice were given an intravenous injection of 100 �g of DNP-HAS (Biosearch

echnologies, Novato, CA, USA) in 1% Evans blue dye (Sigma–Aldrich) [21]. Thirtyinutes later, mice ears were cut into pieces and soaked overnight in 1.5 ml acetoneith 0.5% sodium sulfate, and extravasated blue dye quantified at 620 nm by a spec-

rophotometer (UV-2102C, Unico Instrument, Shanghai, China). To measure mAAFPction duration, mice were injected with mAAFP (6.13 mg/kg) 24 days before sen-itization with mouse IgE anti-dinitrophenyl (DNP) and challenge with DNP-HAS

istry 46 (2011) 858–863 859

as described above. Optical density (OD620) of extracted blue dye was calculatedas follows: OD620 sample = OD620 right ear − OD620 left ear. The inhibition efficiency ofmAAFP on inhibiting allergy reaction was calculated as follows: inhibition efficiency(%) = [(OD62 sample − OD620 control)/OD620 control] × 100.

3. Results

3.1. Transient transfection in 6-well plates

Before conducted in bioreactor, parameters involved in TGEprocess were optimized in 6-well plates, including DNA carrier,transfection time, preparation of DNA/PEI coprecipitation, DNAdosage, DNA/PEI ratio, and cell density at the time of transfection.

Both branched and linear 25 kDa PEIs were reportedly used inTGE as DNA carrier [9,15,22]. Our preliminary experiments showedthe linear PEI was better than the branched one in terms of bothtransfection efficiency and the level of mAAFP expression (datanot shown). Furthermore, most of serum-free media were unfa-vorable to transient transfection although they were beneficial tocell growth. In our study, the CDAGT-CHO medium was found trans-fectable, but led to cells clumping at low viability and subsequentlow productivity (data not shown). In order to solve this problem,one volume of PF-CHO medium was topped up to the culture at6 h post-transfection and gave a high expression level (data notshown). Therefore, the combination of CDAGT-CHO medium andPF-CHO medium was used for transient transfection in this work.

The preparation of DNA/PEI coprecipitation is another key stepin TGE. To establish a good scheme, six different media were usedfor DNA/PEI coprecipitation, which were 5% glucose, 0.9% NaCl, H2O(pH 7.0), DMEM/F12, PBS (pH 7.2) and CDAGT-CHO. The highesttransfection efficiency (36%) and mAAFP expression level (3.4 mg/l)were achieved with DNA and PEI coprecipitated in DMEM/F12(Fig. 1a). For reaction time, DNA and PEI were mixed and incu-bated ranging from 5 to 30 min before transfection. It was foundthat 5 min incubation gave the highest transfection efficiency (35%)and mAAFP expression level (2.9 mg/l) (Fig. 1b). Then, the reactionvolume with DMEM/F12 ranging from 5% to 30% of the culture vol-ume were compared, showing that DNA/PEI complex prepared in20% of the culture volume led to the highest transfection efficiency(35%) and mAAFP expression level (3.2 mg/l) (Fig. 1c).

DNA dosage (a certain amount of DNA to transfect a certain num-ber of cells) and DNA/PEI ratio can also greatly affect transfectionefficiency in TGE [14,23,24]. To optimize the DNA dosage in our sys-tem, the transient transfections were conducted with DNA dosageranging from 1 �g/106 cells to 6 �g/106 cells at the DNA:PEI ratioof 1:2. The results showed that both transfection efficiency andmAAFP expression level increased with DNA dosage varied from1 �g/106 cells to 4 �g/106 cells (Fig. 1d). Then, DNA/PEI ratios rang-ing from 1:1 to 1:4 were evaluated at DNA dosage of 4 �g/106 cells.As shown in Fig. 1e, the highest transfection efficiency (29%) andmAAFP expression level (2.8 mg/l) were achieved at the DNA:PEIratio of 1:2.5. Therefore, the DNA dosage of 4 �g/106 cells and theDNA/PEI ratio of 1:2.5 were applied in bioreactor operation to pro-duce mAAFP.

Finally, the transient transfection conducted at different celldensities of 0.5 × 106, 1 × 106 and 2 × 106 cells/ml showed that thetransfection efficiency was almost the same, but the highest levelof mAAFP expression was yielded with transfection at the densityof 2 × 106 cells/ml (Fig. 1f).

Overall, the optimized linear 25 kDa PEI-mediated TGE systemin CHO-S cells was established as follows: DNA and PEI (DNA/PEI

ratio of 1:2.5) were coprecipitated in DMEM/F12 at 20% of theculture volume with DNA dosage of 4 �g/106 cells. After 5-minincubation, the DNA/PEI complex was added to the culture at adensity of 2 × 106 cells/ml in CDAGT-CHO medium. At 6 h post-transfection, one volume of PF-CHO medium was topped up to

860 L. Cheng et al. / Process Biochemistry 46 (2011) 858–863

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ig. 1. Optimization of TGE parameters in CHO-S cells. DNA/PEI co-precipitates (4–30 min; (c) DMEM/F12, 5–30% volume of cell suspension; (d) DNA dosage from 1:4 at the DNA dosage of 4 �g/106 cells; (f) different cell density, 0.5 × 106, 1.0 × 106

erminate the transfection. This optimized transfection scheme wassed for mAAFP production in bioreactor.

.2. TGE optimization for mAAFP production in bioreactor

The optimized transfection process was conducted in a 1.3 lioreactor as described under Section 2. As shown in Fig. 2, the cellensity reached a maximum of 4 × 106 cells/ml at 3 days post trans-ection (Fig. 2a), and the cell viability gradually decreased to 50% at

days post transfection. After transfection, mAAFP concentrationontinuously increased and reached 2.2 mg/l at the end (Fig. 2d).owever, the total production was only about 2 mg, due to the

hort production duration which was mainly caused by depletionf nutrients.

NA and 8 �g PEI) were prepared in: (a) different media; (b) 100 �l DMEM/F12 for6 cells to 6 �g/106 cells with the DNA/PEI ratio of 1:2; (e) DNA/PEI ratio from 1:1 to106 cells/ml, with the DNA dosage of 4 �g/106 cells and the DNA/PEI ratio of 1:2.5.

To extend the production duration, a fed-batch culture wascarried out after transfection with an enriched feeding mediumcomprising 10× (based on DMEM/F12 formula) amino acids perliter, 90 g/l glucose and 14.5 g/l l-glutamine. The feeding wasstarted as described under Section 2. With this feeding strategy, thepeak cell density reached to 6.1 × 106 cells/ml which was 50% morethan that of the batch culture (Fig. 2a), and the culture durationextended 2 days while cell viability decreased to about 50% (Fig. 2b).The final mAAFP concentration reached to 10 mg/l (Fig. 2d), 3.6-foldincrease compared to the batch culture. Besides, with the increased

culture volume during the feeding process, the total productionincreased to 13 mg, 5.5-fold increase over that of batch culture.

To further extend production duration and improve proteinexpression, mild hypothermic strategy was adopted with tem-perature lowered to 32 ◦C when the cell density reached to

L. Cheng et al. / Process Biochemistry 46 (2011) 858–863 861

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tyrosine by Fc�RI-associated Lyn and inhibition of Fc�RI signaling[25]. GE2, taking the advantage of this Fc�RII negative signal-

ig. 2. TGE of mAAFP in 1.3 l bioreactor: � batch, � fed-batch, � hypothermic fed-bepeated three times, and one representative set was given.

× 106 cells/ml at 3 days post-transfection. The result showed thathe cell viability was maintained above 50% for 11 days (Fig. 2b),

days more than that of the batch culture. In addition, the finalAAFP concentration (20 mg/l) increased 8 fold (Fig. 2d) and the

otal mAAFP production (30 mg) increased 14 folds.

.3. mAAFP efficacy in passive cutaneous anaphylaxis

To test mAAFP efficacy in vivo, 20 Kun-ming mice were ran-omly divided into four groups and given different dosages ofmg/kg, 0.68 mg/kg, 2.0 mg/kg and 6.13 mg/kg, respectively. As

hown in Fig. 3, at dosage of 6.13 and 2.0 mg/kg, mAAFP was effi-acious in inhibiting Evans blue dye extravasation, and at a dosagef 0.68 mg/kg, mAAFP exhibited partial efficacy. Moreover, threether mice were treated with mAAFP (6.13 mg/kg) and then waited

ig. 3. Efficacy assay of mAAFP to inhibit allergy in mouse PCA model. Mice werenjected subcutaneously with mAAFP at different dosages on the back neck 1 dayefore sensitization with mouse IgE anti-DNP on the ear. Four hours later, the miceere challenged intravenously with DNP-HAS plus 1% Evans blue dye. The ears were

emoved 30 min after challenge, soaked overnight in acetone with 0.5% sodium sul-ate, and the extracted blue dye was spectrophotometrically measured. The resultsere repeated in 3 independent experiments. Error bars showed the standard devi-

tion calculated from individual mice in each experiment.

(a) Cell density; (b) viability; (c) residual glucose; (d) mAAFP. The experiment was

24 days before sensitization with anti-DNP IgE and challenge withthe allergen DNP-HSA. It was found that mAAFP was still efficacious(95%) (Fig. 4), indicating that the mAAFP efficacy could last at least24 days upon administration in mouse.

4. Discussion

Coaggregation of Fc�RI with Fc�RII on mast cells and basophilsleads to the rapid tyrosine phosphorylation of the Fc�RII ITIM

ing in human mast cells and basophils, has a great potential forallergic diseases therapy [26]. AAFP is another Fc�∼Fc� fusion pro-tein, whose structure was CH�2–CH�3–CH�4–�hinge–CH�2–CH�3,

Fig. 4. The duration of mAAFP efficacy in vivo. Six Kun-ming mice were randomlydivided into two groups and injected subcutaneously with mAAFP at dosage of6.13 mg/kg on the back neck 1 day and 24 days before sensitization with mouseIgE anti-DNP on the ear. Three other mice not receiving mAAFP were set as control.Four hours later, the mice were challenged intravenously with DNP-HAS plus 1%Evans blue dye. The ears were removed 30 min after challenge, soaked overnightin acetone with 0.5% sodium sulfate, and the extracted blue dye was spectropho-tometrically measured. The results were repeated in 3 independent experiments.Error bars showed the standard deviation calculated from individual mice in eachexperiment.

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62 L. Cheng et al. / Process Bi

ithout any non-human-origin sequence. AAFP may be moreromising due to less immunity when used in human if the flexibleinge acts as the linker in GE2 enabling Fc� and Fc� to simultane-usly bind with their individual receptors. Moreover, it is difficulto evaluate efficacy of AAFP on human cells due to limitation of

odels. In this work, we attempted to establish an optimized tran-ient gene expression system to produce a mouse homolog of AAFPnd then test its efficacy in allergy therapy in mouse.

As a fast protein expression system, transient gene expressionTGE) has been extensively studied and optimized by many groupsrom both industry and academy [5,8,14,27]. However, it is com-

on to see conflicting data reported, indicating the difficulty ineveloping a universal process. Hence, in-house TGE process has toe established and optimized before applying it to produce mAAFP

n bioreactor.CDAGT-CHO medium showed satisfactory transfection efficiency

ut poor final product titer partially because of the short pro-uction duration and low cell viability. The low productivity inDAGT-CHO medium was also reported by Ye et al. [9]. On thether hand, the protein production could be significantly improvedhen PF-CHO medium was topped up to the culture after trans-

ection. The combined use of CDAGT-CHO and PF-CHO media waseported for transient transfection for the first time. Afterwards,ll other parameters which might affect transfection efficiency androtein expression were systematically optimized. However, withhis optimized TGE process, only 2 mg mAAFP was produced in a.3 l bioreactor in a batch culture. Therefore, the optimization ofransfection to achieve a high transfection efficiency is not enougho increase the yield. Then, fed-batch with mild hypothermia wasdopted to provide sufficient nutrition for cell growth and extendhe production duration. Finally, the culture duration was extendedrom 5 days to 11 days, and the total mAAFP production was reachedo 30 mg which was 14 folds higher than that of batch culture. Theseesults demonstrated that recombinant protein production coulde greatly enhanced by controlling the post-transfection process inGE. In this study, we succeeded in producing about 30 mg mAAFPn <3 months.

Passive cutaneous anaphylaxis (PCA) is a classical model forllergen test as well as efficacy evaluation of an anti-allergy drug.n this work, the PCA results demonstrated that mAAFP was effica-ious in inhibiting IgE-mediated type I hypersensitivity reactionsn mice, and the inhibition efficiency could reach 96%. Importantly,

ith mAAFP administration at a dosage of 6.13 mg/kg, the inhibi-ion efficiency could still remain 95% after 24 days. Generally, theerum half-life of IgG1 was only 21–23 days and that of IgE wasven shorter [28,29]. In another study, we found that the humanAFP only has a serum half-life of 1.5 days in monkeys (data nothown). These results indicated that once mAAFP bound to mastells or basophils after administration, the clearance rate of mAAFPould be greatly reduced and the serum half-life of mAAFP could

ast longer than that of IgG1. All these results suggested that AAFPas potential to completely protect people from allergy by severaloses a year.

Although there is no synthetic linker in mAAFP, it is able tofficaciously inhibit allergy in PCA model. This suggests that theexible IgG1 hinge can act as the linker in GE2 and cause AAFP tooaggregate Fc�RI with Fc�RII, and thus inhibit allergic reaction.owever, the mechanism of inhibition by coaggregation of Fc�RIith Fc�RII was still needed to be studied by assaying the key inter-ediate signaling molecules in the reaction. Here, there is no doubt

hat AAFP has very strong anti-allergy potential as shown in mouse

CA model in this work. Since no non-human-origin sequence isnvolved in the molecule, AAFP should have less immunity and cane used more favorable in human compared with GE2.

In summary, we developed an optimal transient transfectionrotocol for CHO-based TGE to produce a mouse homology of AAFP

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istry 46 (2011) 858–863

(mAAFP). Compared with the traditional batch in TGE process, fed-batch culture was found to increase the production level 3.5-fold.Moreover, combined use of fed-batch and mild hypothermia led toa 8-fold increase in expression level of mAAFP from 2.2 to 20 mg/l.The classical PCA model was applied for function assay of mAAFP,and the efficiency of mAAFP in inhibiting allergic reaction could be96%. Beyond our expectation, 95% inhibition efficiency could still beobserved at 24 days after mAAFP administration. This work demon-strated that the IgG1 hinge enabled AAFP to coaggregate Fc�RI withFc�RII, and subsequently activated the inhibitory signaling path-way of Fc�RII. Without non-human-origin sequence in its moleculestructure, AAFP might act as a strong and durable anti-allergy drugin human.

Acknowledgements

This work was supported by grants from the Open Project Fund-ing of State Key Laboratory of Bioreactor Engineering of China andNational Special Project of Key Technology of China (2009ZX09503-013-2 and 2009ZX09102-249).

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