IRES-mediated Tricistronic vectors for enhancing generation of high monoclonal antibody expressing CHO cell lines

Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells

“…3 Poor coupling of the mAb and selection marker genes for such designs results in a significant proportion of non-expressing clones surviving drug selection. [4][5][6] Another disadvantage of having separate expression units is the lack of accurate control of the relative LC and HC expression levels. Varied LC:HC ratios had been reported for clones generated using both co-transfection vectors and multi-promoter single vectors.…”

“…Varied LC:HC ratios had been reported for clones generated using both co-transfection vectors and multi-promoter single vectors. [6][7][8][9] The ratio of LC:HC expression affects both mAb expression level and quality, such as aggregation and glycosylation. [8][9][10][11][12][13][14][15][16][17] Tricistronic vectors using internal ribosome entry sites (IRES) or 2A peptides to express the LC, HC and a selection marker gene in one transcript provides accurate control of the relative expression of LC over HC.…”

“…[8][9][10][11][12][13][14][15][16][17] Tricistronic vectors using internal ribosome entry sites (IRES) or 2A peptides to express the LC, HC and a selection marker gene in one transcript provides accurate control of the relative expression of LC over HC. 6 Expressing the LC, HC and selection marker genes on one transcript also minimizes non-expressing clones because none of the 3 genes will be expressed should the vector be fragmented. 6,18,19 IRES and 2A peptides use different mechanisms for co-expression of multiple genes in one transcript.…”

“…6 Expressing the LC, HC and selection marker genes on one transcript also minimizes non-expressing clones because none of the 3 genes will be expressed should the vector be fragmented. 6,18,19 IRES and 2A peptides use different mechanisms for co-expression of multiple genes in one transcript. When using IRES to express multiple genes in one mRNA, the gene directly downstream of the promoter is translated by the canonical cap-dependent mechanism, and those downstream of IRES are translated by a cap-independent mechanism.…”

“…Because the cap-independent mechanism has lower translation efficiency than the cap-dependent mechanism, 20 co-expressing LC and HC using IRES will result in unbalanced expression with lower expression of the IRES-driven gene. 6,15,18,21,22 In contrast, 2A linked genes are translated in one open reading frame and "self-cleavage" occurs co-translationally to give equal amounts of the co-expressed proteins. [23][24][25] Many 2A peptides have been identified from viruses.…”