Detection of purified antibody was performed with FITC-conjugated anti-porcine IgA (a+c) or with FITC-conjugated anti-FedF (b). antigen could be produced in CHO-K1 cells. After transfection, a strong correlation was found between antibody production and GFP expression (r = 0.69) using image analysis of formed monolayer Imperatorin patches. This Imperatorin enables the rapid selection of GFP-positive clones using automated image analysis for the selection of high producer clones. This vector design allowed the quick selection of high producer clones within a time-frame of 4 weeks after transfection. The highest producing clone experienced a specific antibody productivity of 2.32 pg/cell/day. Concentrations of 34 mg/L were obtained using shake-flask batch culture. The produced recombinant antibody showed stable expression, Cbll1 binding and minimal degradation. In the future, this antibody will be assessed for its effectiveness as an oral vaccine antigen. KEYWORDS:CHO, recombinant antibody, chimeric, 2A peptide, production, expression, GFP, FACS, antibody engineering == Introduction == Systemic vaccines often fail to induce an effective mucosal immune response, characterized by the induction of pathogen-specific secretory immunoglobulin A (SIgA).1Oral vaccines are much more effective in achieving mucosal immunity and have the Imperatorin added benefit of being easy and safe to administer.2One of the main drawbacks of oral vaccination is the poor uptake by the intestinal epithelium and the ensuing delivery to the underlying gut-associated lymphoid tissue.3Selective targeting of vaccine antigens to a transport protein around the intestinal epithelium might solve this problem.4Recently, we showed that antibody-mediated delivery of antigens towards aminopeptidase N (APN), a membrane receptor expressed on enterocytes and involved in epithelial transcytosis, triggered systemic and mucosal antibody responses in a piglet model.5,6However, in these experiments, porcine APN-specific rabbit or mouse IgG were used, which resulted in rabbit or mouse IgG-specific immune responses upon oral vaccination in piglets. The presence of these antibodies might impact the efficacy of APN targeting in a prime-boost vaccination regime. To minimize these responses, a recombinant porcine APN-specific chimeric mouse-porcine IgA antibody, linked with a clinically relevant antigen, was designed. By replacing the mouse IgG constant domains with porcine IgA, minimal immune response and increased antibody stability is usually expected.7-9 Most recombinant antibodies are produced in Chinese hamster ovary (CHO) cells due to their capacity for correct folding, assembly and glycosylation, leading to improved production. The creation of a stable, high producer cell collection is essential to support the high demand for antibody production.10Antibodies are complex molecules consisting of both heavy and light chain polypeptides. Moreover, the ratio of both chains affects the final production of the complete antibody.11,12Efficient co-expression of the heavy and light chain is therefore one of the most important aspects in monoclonal antibody production. This co-expression can generally be achieved by either co-transfecting two individual vectors, each encoding a single antibody chain, or by transfecting a single vector encoding both chains.13Expression on separate vectors often results in a poor balance of light and heavy chain expression levels, leading to reduced antibody production. Multiple studies have shown that expressing both chains from a single vector significantly enhances the expression ratio.14,15Co-expression on a single vector can be achieved by either using two separate promotors, an internal ribosome access site (IRES) or self-cleaving 2A peptides.16The use of an IRES-element often leads to reduced protein expression of downstream genes, ranging from 6 to 100%, making this system unpredictable.17-19 Self-cleaving 2A peptides are short, highly conserved sequences of 1822 amino acids derived from viruses, such as foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A) and thosea asigna virus (T2A). They mediate cleavage of polypeptides during translation by steric hindrance, resulting in ribosomes skipping the formation of a glycyl-propyl (G-P) peptide bond at the C-terminus of the 2A peptide.20,21After successful skipping, the 2A peptide remains bound to the upstream protein and often a furin cleavage site is inserted to remove the remaining peptides. The use of 2A peptide cleavage mostly prospects to higher expression levels compared to IRES-based expression, 13but can also lead to generation of aggregates due to incorrect cleavage and folding. 16Efficiency of correct cleavage and antibody production is usually highly dependent on the cell collection used and 2A peptide sequence. T2A peptide cleavage in addition to a GSG sequence (GT2A) showed the highest cleavage efficiency and antibody expression levels in CHO cells.20 Another major bottleneck in the production of recombinant antibodies is the selection of stable transfected cells with high expression. By using a 2A peptide sequence to link GFP expression to protein production, the screening time and effort could be significantly improved. Co-expression of fluorochromes.
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