Transient Gene Expression And Mass Preparation Of Mouse-Derived Fusion Protein Fcε-Fcγ And Its Anti-Allergy Efficacy In Mouse

Allergic diseases have reached epidemic proportions worldwide, and its prevalence has increased dramatically as environmental degradation and air pollution within the last few decades. Allergic asthma is one of the most common allergic diseases, especially in industrialized countries, which is considered as one of the four major chronic diseases by the World Health Organization. People have been trying to find safe and effective treatments for asthma, and traditional drug-based improvement and optimization are gradually replaced by the discovery of new therapeutic targets and the development of biopharmaceuticals. Nowadays, there are several biopharmaceuticals for asthma treatment in clinical studies, and more candidates in pre-clinical development. Blocking the binding of an IgE multivalent antigen to an FcεRI receptor on the surface of mast cells or basophils to prevent the occurrence of allergic reactions is the main direction of the treatment for allergic diseases.The Fcs-Fcy fusion protein (AAFP) is a promising drug candidate for IgE-mediated allergic diseases by bridging FcεRI and FcyRII together on mast cells and basophils and subsquently initiate the inhibitory signaling by FcyRII. The coaggregation depends on that the flexible IgGl hinge in AAFP allows a necessary spatial relationship between AAFP and its two receptors. Moreover, it is difficult to evaluate efficacy of AAFP on human cells due to limitation of models. In this work, we aimed to establish an optimized transient gene expression system to produce a mouse homolog of AAFP and then test its efficacy in allergy therapy in mouse.As a fast protein expression system, transient gene expression (TGE) has been extensively studied and optimized by many groups. However, the process is protein-dependent, in-house TGE process has to be established and optimized before applying it to produce mAAFP in bioreactor. The TGE process is rather complicated and its efficiency is affected by many factors, including host cell, DNA carrier, serum-free medium and transfection process. CHO cells have become more favorable at present because a large number of recombinant proteins produced for clinical trials were expressed in stably transfected CHO cells. To avoid the variation in the recombinant protein derived from different host species, we have to expend much effort to optimize CHO-based TGE.The TGE process was established for the first time with the combination of CDAGT-CHO and PF-CHO media, the former for transfection to give high transfection efficiency, and the latter for expression to give high mAAFP titer. All parameters which might affect transfection efficiency and protein expression were systematically optimized. Firstly, two different expression vectors pCI-EG and pID-EG were transiently transfected in CHO-S cells. A better level of expression was reached when using pID-EG as an expression vector compared with pCI-EG. Then other parameters involved in TGE process were optimized in 6-well plates, including DNA carrier, transfection time, preparation of DNA/PEI coprecipitate, DNA dosage. DNA/PEI ratio, and cell density at the time of transfection. Overall, the optimized linear 25 kDa PEI-mediated TGE system in 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 the culture volume with DNA dosage of 4μg/106 cells. After 5 min incubation, the DNA/PEI coprecipitate was added to the culture at a density of 2×106 cells/ml in CDAGT-CHO medium. At 6 h post-transfection, one volume of PF-CHO medium was topped up to terminate the transfection. This optimized transfection scheme was used for mAAFP production in bioreactor.However, with this optimized TGE process, only 2 mg mAAFP was produced in a 1.3 L bioreactor in a batch culture. After the optimization of transfection aiming to achieve high transfection efficiency, the increase of expression yield was concerned. Therefore, feeding and mild hypothermia were adopted together to provide sufficient nutrition for cell growth and extend the production duration. Finally, the duration of culture was increased from 5 days to 11 days with the cell viability maintaining at above 50%. The total mAAFP production was reached to 30 mg, which was 14 folds higher than that of batch culture. These results demonstrated that recombinant protein production could be greatly enhanced by controlling the post-transfection stage in TGE. Then, the TGE process was conducted in 5 L bioreactor and a high expression level of 25 mg/L was achieved. mAAFP was subsequently purified by rProteinA Sepharose Fast Flow and the purity reached 98% with the recovery of more than 85%. The obtained mAAFP was identified by SDS-PAGE and Western blot.Passive cutaneous anaphylaxis (PCA) is a classical model for allergen test as well as efficacy evaluation of an anti-allergy drug. In this work, the PCA results demonstrated that mAAFP was efficacious in inhibiting IgE-mediated type I hypersensitivity reactions in mouse, and the inhibition efficiency reached 96%. Importantly, with mAAFP administration at a dosage of 6.13 mg/kg, the inhibition efficiency could still remain 95% after 24 days. These results indicated that once AAFP binded to mast cells or basophils after administration, the clearance rate of mAAFP would be greatly reduced and its serum half-life span could last much longer than that of IgGl. This work shows that AAFP has great potential for allergy therapy, and lays the solid basis to detail mechanism and efficacy of AAFP on human cells for the therapy of allergic diseases.