Most of the therapeutic antibodies are produced in mammalian cells, as the post translational components, folding and secretion of these cells are appropriate for generating antibodies similar to those native to human beings. Mammalian cell production of antibody is suitable for transient expression than in cell lines. Human embryonic kidney cell lines or HEK-293 cell lines are found to be efficient in the transient expression of proteins as they are known to be easily transfected with plasmid DNA. HEK-293 cell lines are created by transforming the embryonic tissue with sheared adenovirus 5 DNA. These cells transformed by SV40 virus are called as HEK-293T cells and those transformed by Epstein Barr virus (EBV) are called as HEK-293E cells. The transfection of plasmid DNA into HEK-293 cells was carried out by calcium phosphate, polymers and cationic liposomes. The polyethyleneimine (PEI) enables efficient plasmid insertion with low cytotoxicity.
This study focused on optimizing the vectors involved in mammalian cell expression for cloning scFv fragments out of entire antibody phage display libraries into IgG like scFv-Fc format. The researchers in this study have demonstrated that transient production of scFv-Fc in HEK-293-6E cells is efficient and versatile.
Results of the study
Transient expression of 14 mg/L of scFv-Fc and 7 mg/L of immunoRNase was initially done in HEK-293 cell lines using pCMV vectors. The yield of these proteins in HEK-293T cell lines was between 4 and 6mg/L. To clone a single chain antibody fragment in scFv-Fc format, a vector was constructed called pCMV-hIgG1Fc-XP. This construct was the resultant of pCMV-HC modified at 5' untranslated region by adding a multiple cloning site compatible to Nco/NotI. Vectors having Nco/NotI site are added with "XP" in their names. The above construct was further optimized at the region of 3' stop translation sequences present in the Fc gene fragment.
The pCMV3.2-hIgG1Fc-XP plasmid was constructed with the deletion of 3' terminal lysine codon and pCMV4.2-hIgG1Fc-XP was constructed with the introduction of BamHI site reconstruction codons. The vectors pCMV2.1, pCMV2.2, 2.3, 2.4, 2.5, pCMV3.2 and 4.2 could show 1.5 to 3 times higher transient expression of scFv-Fc antibody in HEK-293T cells. The vector pCMV2.5-hIgG1Fc-XP shows further improvement to avoid unspecific binding of cysteine that usually forms interchain disulfide bonds in between light and heavy chains.
Transient expression of mammalian expression vectors in HEK-293-6E cells
The standard transfection in HEK-293-6E cells using PEI and mammalian expression vectors like pCMV-scFv-hIgG1Fc-4E3 and pCMV-sc-Fv-hIgG1Fc-RNase-4E3 could yield 30 - 40 mg/L of scFv-Fc-RNase and 70 - 80 mg/L of scFv-Fc, after 3 days of production. The volumetric yield of scFv-Fc-RNase was 60 to 70mg/L and of scFv-Fc was 120 to 140mg/L after 5 days of production.
Vector optimization for transient expression in HEK-293-6E cells
The vectors with selection marker and SV40promoter/ori were constructed as scFv-Fc-RNase and pCMV-neo-scFv-Fc. The vectors constructed with EBV oriP were pCMV-oriP1-scFv-Fc-RNase and pCMV-oriP1-scFv-Fc. The pCMV-neo vectors could yield 2.5 times higher expression of scFv-Fc and scFv-Fc RNase than the usual pCMV vectors. The pCMV-oriP1 vectors could yield 3.5 times higher expression of antibody fragment. The supernatants were purified by protein A affinity chromatography and the recovery rates were 80 percent, while the recovery rate was 50percent before purification. The efficiency of transfection examined by co-transfection along with a fluorescent reporter plasmid was 75 to 90 percent.
Optimization in the production of scFv-Fc and immunoRNase in HEK-293-6E cells
Higher production levels were observed after 6 to 7 days by the vector having pCMV-oriP1 with the yield of 450mg/L and the vector expressed scFv-Fc-4E3 and scFv-Fc-RNase-4E3.The supplementation of glucose with VPA (histone deacetylase inhibitor) could increase the production of scFv-Fc-RNase in two samples while glucose alone could not yield any higher results. The treatment of medium with VPA will reduce the cell growth and elevate the cell concentration to 4X106cells/mL, after six days of transfection. The other two independent groups have increased concentration of the cells as 6 - 8X 106cells/mL. The viability of the cells began gradually to reduce after 7 days of production in other groups while in VPA treated group, viability remained greater than 90percent.
An optimized library compatible scFv-Fc vector was generated to express HEK-293-6E cells. The plasmid construct of pCMX2.5-hIgG1Fc-XP having CMV promoter was embedded with scFv-Fc-4E3 antibody and scFv-Fc-RNase-4E3 immunoRNase. The resultant plasmids obtained for transfection into HEK-293-6E cells are pCSE-scFv-hIgG1Fc-RNase-4E3 and pCSE-scFv-hIgG1Fc-4E3. Transient production of these vectors in HEK-293-6E cells could yield 300mg/L of scFv-Fc RNase and 400mg/L of scFv-Fc, which were higher compared to the production of vectors with pCMV-oriP1. The production was higher after 6 days, while the cell viability was greater than 90 percent by then.
Transient production of IgG like scFv-hIgG1Fc antibodies by the pCSE2.5-hIgG1Fc-XP vector sub cloned in-frame of the Fc gene with more than 20scFv gene fragments resulted in the volumetric yields of scFv-Fc antibodies as 100 to 600mg/L. The hIgG1Fc antibody fragment without scFv was produced at 900mg/L. The scFv-Fc antibodies were purified and stored for six months at the temperature of four degree centigrade in PBS and without any protein mixed into it. There was no proteolytic degradation observed.
Volker Jager, Konrad Bussow, Andreas Wagner, Susanne Weber, Michael Hust, Andre Frenzel, and Thomas Schirrmann. High level transient production of recombinant antibodies and antibody fusion
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