| Objective:Acute myeloid leukemia (AML) is a heterogeneous disease, treatment failure and relapse is the major cause for poor prognosis. Leukemic stem cells (LSCs) is the origin of disease persistence, drug resistance and relapse, so the LSCs targeted therapy may present as a curable treatment for AML. The signal transduction drived from interleukin-3 receptor involved in the stimulation of proliferation of AML blasts, and interleukin-3 receptor a chain (IL3RA)/CD123 is a marker for LSCs in AML, at variance with normal hematopoietic stem cells (HSCs) that are IL3RA-. So, the LSCs targeted, anti-CD123 antagonist monoclonal antibody may present as a promising therapy for AML.Methods:Partâ… :To product and purify IL3RA ECD/Fc fusion protein as an immunogen, we cloned the extracellular domain (ECD) of CD123 to human IgG Fc and constructed two eukaryotic expression vectors, pG4Fc-3.3-hIL3RA SP+ECD and pYDll-hIL3RA ECD, then HEK-293F cells were transfected with these plasmids transiently after optimizing of the transfection conditions, finally we purified and characterized the generated fusion protein. On the other hand, we cloned coding sequence (CDS) of CD123 to construct the other two vectors, hIL3RA CDS-pEGFP-N1 and pcDNA3.1(+)-IL3RA CDS. The former will be used to generate a CD123 overexpression cell line for subsequent antibody screening; the later will be used as immunogen. An adenovirus for immunization boost is needed for the immunization with plsmid.Part II:To generate a CD123 overexpression cell line for antibody screeing, we transfected CHO cells with hIL3RA CDS-pEGFP-Nl and subjected to FACS sorting and G418 screening. The expression of generated cell lines were evaluated by FACS, Elisa and Western blot. At the same time, we immunized Balb/c mice with different methods, including IL3RA ECD/Fc fusion protein, CD123-expressing TF-1 AML cell line and eukaryotic vectors pcDNA3.1(+)-IL3RA CDS. After screening by Elisa, spleen cells of immunized mice with high serum titer were fused with myeloma SP2/0 cells. The positive wells were picked out by Elisa and indirect FACS. After several round of limited dilution and screening, hybridoma cell lines capable of secreting anti-CD123 antibodies were generated for subsequent functional assays.Part III:To evaluate the ability of generated hybridoma cell lines to secret antibodies and block the biding of IL-3/IL3RA and the following proliferation effect competitively, we established IL-3 antagonist assay. We also established several acute leukemia NOD/SCID xenotransplantation models to test whether these antibodies could inhibit the homing of CD34+CD38- and the engraftment of CD45+ leukemic cells, which will reduce tumor burden and improve survival.Results:Although immunization with TF-1 cells generated no antibodies, we still obtained 3 and 8 hybridoma cell lines by immunization with fusion protein and plasmid, respectively. Determined by Elisa and indirect FACS, all of the antibodies secreted from these 11 cell lines had affinity to IL3RA ECD/Fc and CD123 expressed on TF-1 and 8G9 cells, especially, one of them was confirmed as an antagonist antibody to block the biding of IL-3/IL3RA and the following proliferation effect. We also established several acute leukemia NOD/SCID xenotransplantation models to test the anti-cancer property of these antibodies.Conclusion:Immunized with plasmid and boosted with adenovirus is a very efficient way to gain therapeutic antibody with certain function. In the 11 hybridoma cell lines capable of secreting anti-CD123 antibody, one named 5A2 was picked out for its ability to block the biding of IL-3/IL3RA and the subsequent proliferation effect competitively. The remaining work is to evaluate whether 5A2 could evoke ADCC to inhibit the homing of CD34+CD38-and the engraftment of CD45+ leukemic cells, which will decrease tumor burden and improve survival. |
PDF Full Text Request