Screening And Expression Of Anti-PD-L1 Monoclonal Drugs

Tumor immunotherapy is a research hotspot in recent years.Anti-tumor therapy drugs targeting PD-1/PD-L1 have brought clinical benefits to many patients with malignant tumors.Programmed death ligand 1(PD-L1)is a protein expressed on the surface of various tumor cells and can combine with programmed death 1(PD-1)on the surface of T cells.In vivo,binding of receptors to ligands inhibits downstream NF-?B transcription and promotes down-regulation of interferon-? secretion,ultimately inhibiting T-cell immunity,leading to the formation of an immunosuppressive tumor microenvironment,and allowing tumor cells to escape from the body's immune system monitoring and killing.Blocking the PD-1/PD-L1 signaling pathway can reverse the tumor immune microenvironment and restore the anti-tumor activity of T cells,thereby enhancing endogenous anti-tumor immune effects.The purpose of this study was to humanize PD-L1 antibodies from mice to produce highly effective antibody drugs.In order to obtain humanized anti-PD-L1 antibody sequences,the light and heavy chains of the resulting anti-PD-L1 antibodies were humanized by Ig BLAST to obtain an optimized antibody template.The pcDNA3.1 expression vector was constructed.The expression vector was transfected into HEK-293 cells by transient transfection and the 17 humanized antibodies were correctly expressed by SDS-PAGE.The indirect ELISA method was established for detecting the biological activity of antibodies.The optimal antigen coating concentration for indirect ELISA was 0.25 ?g/ml,the beginninging concentration of anti-PD-L1 antibody standard was 0.25 ?g/ml,and the optimal concentration of blocking solution was 2% BSA,the optimal closure time was 2 h.The optimal dilution for the secondary antibody is 1:4 000.Anti-PD-L1 antibody sample 1 had a titer of 125,a concentration of 66.66 ng/ml and a sensitivity of 6.3 ng/ml;sample 2 had a titer of 125,a concentration of 81.8 ng/ml,and a sensitivity of 5.9 ng./ml.After statistical analysis of the intra-and inter-assay reproducibility tests for samples 1 and 2,the coefficient of variation was <10%.According to the data,this ELISA method is suitable for the detection and screening of anti-PD-L1 antibodies.Using the above methods for expression,the collected samples were screened for activity screening,and humanized anti-PD-L1 monoclonal antibody combinations with strong affinity and expression in HEK-293 cells were selected: 1Z2 Q,1Z6Q,3Z2 Q,3Z6Q,4Z6 Q.The humanized mouse anti-PD-L1 antibody was initially successfully selected and the bioactive humanized antibody 5 group was screened,which has a certain significance for the further study of the humanized PD-L1 monoclonal antibody.