Background:Reduction/alkylation is one of the leading strategies for the development of antibody drug conjugates (ADCs). Precise control of the reduction process would not only yield a defined number of free thiols per antibody but also result in development of more homogenous conjugates.Methods:In the present study, we investigated the effect of various dithiothreitol (DTT) concentrations, temperature conditions, and DTT exposure times on antibody reduction. After antibody reduction, the Ellman’s test and SDS-PAGE analysis were used to evaluate free thiols produced and confirm the reduction process, respectively.Results:DTT concentration seems to be a potential factor in the reduction process. Concentrations of 0.1, 1, 5, 10, 20, 50, and 100 mM DTT at 37°C for 30 minutes resulted in approximately 0.4, 1.2, 5.4, 7, 8, 8, and 8 thiols per antibody, respectively.Conclusion:Optimized site-specific conjugation can provide better process control and reproducibility for the development of disulfide-based ADCs.
Acute myeloid leukemia (AML) is a clonal hematologic malignancy arising from a small population of leukemic cells initiating the disease. CD123 is differentially expressed in AML blasts compared with normal hematopoietic stem and progenitor cells. The aim of this study was to develop specific monoclonal antibodies (mAbs) directed against AML. Three BALB/c mice were immunized with the human CD123 antigen, and the immune spleen cells were fused with the SP2/0 myeloma cell line. Hybridomas were screened by indirect enzyme-linked immunosorbent assay (ELISA), and the positive hybrids were cloned by limiting dilution. The mAb isotype was determined, ascitic fluids were produced, and antibodies were purified using Fast protein liquid chromatography (Sephacryl S-200). The specificity of the hybridomas was examined by ELISA, cell-based ELISA, and flow cytometry. After three rounds of cell cloning, four anti-CD123 secreting hybridomas were obtained with the IgM isotype. Among them, one stable hybrid, designated sC1, exhibited the higher ability to recognize the CD123 antigen, as compared with the other hybridomas. Our results showed that sC1 has the ability to bind specifically to the CD123 antigen (41.36%) on the cell surface. The anti-CD123 mAb produced in this study may be useful for the development of both diagnostic and therapeutic purposes for AML.
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