Influence on expression of humanized anti-EGFR with different copy numbers of the light chain which site-specific integrated into CHO-K1bak-/bax- cells

• 1、School of Pharmacy,Jiangnan University,Wuxi 214122

Abstract：Objective In order to compare the influence of the expression on copy numbers of the light chain, the genes of humanized anti-epidermal growth factor receptor (anti-EGFR) were site-specific integrated at NW_003626341.1 site of the LOC103162981 gene in anti-apoptotic CHO-K1bak-/bax-(CHO-Ie3) cells with different copy numbers of the light chain through CRISPR/Cas9 gene-editing technology. Methods A pEF1α-LH plasmid and a pEF1α-LLH plasmid contained genes encoding the light and heavy chains of anti-EGFR, the enhanced green fluorescent protein (EGFP) were respectively co-transfected with the single guide RNA (sgRNA) plasmid and Cas9 plasmid to CHO-Ie3 cells with mediation of LipofectamineTM 3000 Kit. After selecting and sorting, monoclonal cell lines were generated in a 96-well plate. Dolt blot, PCR cloning and Western blot were used to verify whether genes of interests were integrated and expressed accurately in the expect locus. The expression of anti-EGFR was analyzed through batch culture. Results Two CHO-Ie3-LLH cell lines and one CHO-Ie3-LH cell line correctly expressed anti-EGFR via screening and verification. The probability of the light chain with double-copy undergoing site-specific integration is about 3.51%, while the light chain with single-copy is about 1.89%. The antibody produced by CHO-Ie3-LLH cell lines were 110.13 mg/L and the expression level is increased by 35.32 % compared with CHO-Ie3-LH cell lines. Conclusion Humanized anti-EGFR was accurately expressed in CHO-Ie3 cells through CRISPR/Cas9 gene-editing technology. The increase in the copy numbers of the antibody light chain showed a significant effect on the expression of exogenous antibody.

Keywords： site-specific integration anti-Epidermal Growth Factor Receptor CHO cells monoclonal antibodies