A Method For Detecting The Biological Activity Of A Bispecific Antibody PD-L1/CD3-BsAb Targeting CD

Objective: To construct a biologically active reporter gene assay for the bispecific antibody PD-L1/CD3-Bs Ab targeting CD3,and to optimize and validate the method.Methods: PBDP-NFAT-Fluc plasmid was constructed by PBDP transposase system,Jurkat cells were transfected using liposome transfection,and screened by adding 0.8μg/m L of puromycin(for a total of 15 days,with fluid changes every 4-5 days),and the cell line with the highest GFP expression was selected using flow cytometry to construct a Jurkat-NFAT-Fluc cell line.The BS006 virus enters Vero cells through cell surface molecular receptors and performs DNA replication,transcription,translation and viral assembly inside the tumor cells to secrete its encoded protein products to the extracellular.After co-incubation of the sample to be examined(i.e.culture supernatant obtained from Vero cells infected with BS006 virus to be examined)with CD3-expressing Jurkat-NFAT-Fluc cell line and PD-L1-expressing A375 cells,PD-L1/CD3-Bs Ab activates NFAT regulatory elements to initiate and promote downstream Fluc gene expression,therefore,by detecting NFAT Therefore,a standard curve of PD-L1/CD3-Bs Ab fluorescence value versus biological activity U/m L was plotted by measuring the expression level of NFAT-induced luciferase Fluc gene,so as to detect the biological activity of PD-L1/CD3-Bs Ab of the test article.Results: The fluorescence intensity of the cells with two different target ratios(1∶2and 1∶4)showed a tendency to increase with the increase of cell density,and the difference between the compound wells was the smallest when the target ratio was 1∶2,so 1∶2 was chosen as the best target ratio.When three incubation times(2,4 and 6h)were selected,the highest fluorescence RLU values were obtained at an incubation time of 6h.The biologics methodology was validated by bioequivalence,accuracy,precision,durability and specificity.Conclusion: A reporter gene assay for the biological activity of a bispecific antibody PD-L1/CD3-Bs Ab targeting CD3 was successfully constructed,and the related optimization and methodological validation were completed.