Investigation On The Site-specific Integration Of Anti-EGFR At The NW＿003626341.1 Site In CHO Cells

Chinese hamster ovary（CHO）cells are the predominant workhorse for the expression of therapeutic proteins.One of the significant technologies among the biopharmaceutical domain is to construct a recombinant CHO cell line that is capable of stably and efficiently expressing exogenous proteins.Previously,our research team found an intragenomic site,which located at the LOC103162981 gene NW＿003626341.1.The enhanced green fluorescent protein （EGFP）and single-chain protein--human serum albumin（HSA）protein constituted with 580amino acid residues can be stably expressed in this site.In this case,genes of a humanized anti-epidermal growth factor receptor（anti-EGFR,150 k Da）with two light chains（LC）and two heavy chains（HC）were integrated into the same site of CHO-K1 cells.To make a supplement-understanding of the adaptability of the therapeutic protein at the NW＿003626341.1 site through analyzing the stability of exogenous genes and the expression of humanized EGFR antibody during cell culture.The key findings of our study follow below:Ⅰ.A p EF1α-HL and a p EF1α-LH donor plasmid was designed and constructed with an NW＿003626341.1 homologous sequence harboring the genes of differentially arranged LC and HC of anti-EGFR as well as the genes of a dual screening label.Green fluorescence and puromycin were site-specific integrated into CHO cells through CRISPR/Cas9 gene-editing technology.The donor plasmid,sg RNA and Cas9 plasmids were co-transfected into CHO-K1cells and BAK^-/BAX^-double knockout CHO-K1 cells（CHO-Ie3）based on homology-directed repair.After screening,flow cytometric sorting and serial verifications,eight cell lines expressing humanized anti-EGFR were obtained,including three CHO-K1-HL and CHO-Ie3-HL cell lines,one CHO-K1-LH and CHO-Ie3-LH cell line,respectively.The editing-event that genes of anti-EGFR accurately knocked into the NW＿003626341.1 site of CHO-K1 cells was about 1.82-5.17%,and that of CHO-Ie3 cells was about 1.89-4.69%.The obtained recombinant cell lines were all heterozygote.Ⅱ.Three CHO-K1-HL cell lines successfully acclimated in suspension and were subjected to continuous culture for 50 passages.The green fluorescence can be clearly observed in the different passages of three cell lines,and the average fluorescence intensity nearly identical.Among different culture passages,humanized anti-EGFR can stably expressed and the expression level is not significantly different.These results not only verified that the NW＿003626341.1 site in the LOC103162981 gene of CHO cells was a high-quality site that can stably express exogenous proteins,but also suggested that this site can express simple single-chain proteins as well as complex four-chains proteins.Thereby,meeting the requirements of stable expression among the establishment from the master cell bank to the production cell bank and the quality for scale-up culture.Ⅲ.One strain of CHO-K1-HL,CHO-K1-LH,CHO-Ie3-HL and CHO-Ie3-LH cells was selected,respectively,to conduct a preliminary fed-batch culture experiment.The cell density of CHO-K1-LH cells was above 1.9×10⁷cells·m L^-1and the antibody production was116.20-143.15 mg·L^-1.Confocal and FACS analysis indicated that humanized anti-EGFR recognized and bound to the EGFR antigen and endocytosed into human skin squamous carcinoma A431 cells.Together,these results illustrated that exogenous genes were able to be site-specific integrated into the NW＿003626341.1 site in the LOC103162981 gene of CHO cells,which possessed the potentiality to stably express different therapeutic proteins.The sequence of LH is conducive to the expression of antibodies at the NW＿003626341.1 site in the CHO genome.