Modification Of CHO Cell Line Using CRISPR/Cas9 Technology To Produce Monoclonal Antibodies With Enhanced ADCC

Background and aim:Monoclonal antibody therapy has dominated a large proportion in pharmacy market over the past decades.Therapeutic monoclonals are increasingly used for treating cancer and autoimmune disorders.In addition,market data is forecasting mAbs would grow with a higher rate than any other therapeutic class.The mechanism of antibodies-activated immune system is complex,mainly including complement-dependent cytotoxicity?CDC?and antibody-dependent cellular cytotoxicity?ADCC?.ADCC is mediated by antibody Fc portion binding to NK cells Fc?R?a receptor.The efficacy of NK cells recruitment and activation is determined by the affinity of IgG Fc for Fc?R?a.And the affinity is related to the presence or absence of fucose on the oligosaccharide of the IgG Fc.Defucosylated N-oligosaccharide attached to Fc at asparagine297 enhances the ADCC activity through improving the affinity binding to Fc?R?a.Mogamulizumab,a defucosylated,humanized,anti-CC chemokine receptor 4 monoclonal antibody,was proved in Japan in March 2012 for treating relapsed orrefractory CCR4-positive T cells leukemia-lymphoma.Removal of fucose has improved ADCC activity than conventional antibodies.By the way,there are six defucosylated mAbs in clinical studies now.Chinese hamster ovary?CHO?cells have became the most widely used industrial mammalian cell line for produced antibodies.Among the therapeutic antibodies launched all over the world,35.5%are produced by CHO cells.However,more than 80%IgG1 produced by wild type CHO cells possesses fucose glycoform.CHO-S cell line is chosen as a model cell in this study because of suspension character and wide application in industry.At present,there are two FUT8 gene modification cell lines created by homologous recombination?HR?and Zinc finger nucleases?ZFNs?respectively in market.Homologous recombination?HR?was less efficient and time-consuming process.Zinc finger nucleases?ZFNs?were extremely expensive and difficult to design.All of these limited their wide application.CRISPR/Cas9?Clustered Regularly Interspaced Short Palindromic Repeats?system only required guide sequence RNA designed,which is ease of customization,higher targeting efficiency and lower the cost.So we use CRISPR/Cas9 technology to obtain a cell line produced defucosylated mAbs with enhanced ADCC.Method:CHO-S cell line is chosen as the model cell to be modified.To get the defucosylated antibody with enhanced in vitro ADCC activity,we choose exon 9 of FUT8 gene as the target gene to design sgRNA.PX330plasmids transfect into CHO-S cells after sgRNAs inserted.Two rounds of limited dilution were conducted to select the double allele disruptive clones with the assist of lensculinaris agglutinin?LCA?.A stable FUT8^-/-cellclone is obtained.To convince the successful gene completely disruption in CHO-S cells,DNA gel electrophoresis,geneand amino acid sequencing alignments,FITC-LCA membrane binding assay were performed.Then anti-Her2 monoclonal antibody expression vectors were transfected into FUT8^-/-clone.Analysis the N-glycan at 297Asn of antibody with HILIC-HPLC,further convince that the fucose of antibody have been removed completely.Test the antibody affinity to antigen Fc?R?a with BIAcore.Finally,we test in vitro ADCC activity by LDH release kits.Results:FUT8^-/-clone was obtained and convinced by limited dilution,DNA sequencing and FITC-LCA membrane binding test.The FUT8^-/-clone had similar growth profiles compared to wild type CHO-S cells.Monoclonal antibody expressed in FUT8^-/-clones presented no fucose signal by glycan test with HILIC-HPLC assay.The affinity binding to Fc?R?a antigen of antibody expressed by FUT8^-/-clone was 7 times stronger than that produced by wild-type CHO-S cell line with the BIAcore assay.The ADCC activities on SK-BR-3 increased by about 25-fold and on HepG2improved over 8-fold after FUT8 gene was completely disrupted.So we had a conclusion that a stable FUT8^-/-cell line was got through CRISPR/Cas9 system.