CD123 Expression In B-acute Lymphocytic Leukemia And Preparation Of Anti-human CD123 Monoclonal Antibody

Part 1:CD123 expression and significance in pediatric B-acute lymphocytic leukemia patientsObjective: To monitor CD123 expression in pediatric B-acute lymphocytic leukemia(B-ALL) patients and analyze different factors influencing on CD123 expression.Methods:We collected 246 bone marrow samples of children previously in our hospital from May,2013 to December,2014.Of these cases, there were 196 B-ALL patients,22 relapsed B-ALL patients,34 T-ALL patients and 16 healthy children.Applying flow cytometry, we acquired whether CD123 expressed on the surface of these bone samples,analysed mean expressing percentage and level of CD123 in CD123-postive cases.Furthermore,we compared the relationship of the impact of gender,gene,WBC accounts at diagnosis,cytogenetics,maturity of cells with CD123 expression.Results:1.Samples of healthy children did not express CD123,only 8.82% of T-ALLsamples express CD123,while most of B-ALL patients expressed CD123,postive cases accounting for 91.8%.2.CD123 and CD19 were both positive on 91.8% of B-ALL patients.What is more,accompanied by higher CD34 presentation,CD123 showed higher expression.3.The CD123/10/34/19 group could be effectively monitored MRD of 58.01% of B-ALL patients.4.The differences of gender(female or male),age(>10years or<10years),WBC accounts(≧50×109 cells/ml or <50×109 cells/ml)and relapse(yes or no) did not influence the expression of CD123 on B-ALL bone samples(P>0.05).The hyperdiploid B- ALL showed significantly higher CD123 expression than other major subcategories(P<0.001).In contrast, cases withE2A/PBX or TEL/MLLrearrangement had weaker CD123 expression compared with corresponding negative gene groups(P<0.05).Conclusion:1.CD123 could be overexpressed on the majority of B-ALL pediatric patients,thus be applied to monitor minimal residual disease of B-ALL and also be a hopeful target for a novel treatment of relapsed and refractory B-ALL.2.Overexpression of CD123 correlates with the hyperdiploid genotype in B-ALL.E2A/PBX or TEL/AML1 rearrangement correlates with weaker CD123 expression.Part 2:Preparation of a mouse anti-human CD123 monoclonal antibodyPurpose: To develop a novel mouse anti-human CD123 monoclonal antibody,which lays foundation of an insight into the function of human CD123.Methods:We compared CD123 expression levels of several cell lines,from which chose a cell line higher expressing CD123,SHI-1. We used SHI-1 as immunogen to immunize mice three times about every two weeks,then we accquired the immuned spleen cells of one mouse and fuse with meyloma cells SP2/0.Meanwhile we transfected CD123 plasmids into L929 cells to construct a L929-CD123 cell line.Microscopy the condition of hybridoma cells every day,when the hybridoma cells grow up to one tenth of the the hole,we applied flow cytometry to screen hybridoma cells with SHI-1 or L929-CD123 cells by indirect immunofluorescence.Then we used limiting dilution assay to make a monoclonal hybridoma cell, cultured it into cluster,and indetified it by flow cytometry.We injected identified monoclonal hybridoma cells into the enterocoelia of mice.Ascites that mice produced were purified, identified and finally compared with commercial antibody.Results:CD123 plasmids were effectively transfected into L929 cells.By flow cytometry screening,we develop a monoclonal hybridoma,which could secret anti-CD123 antibody,named C1409.Conclusion:We sucessfully accquired a hybridoma cell line stably and persistently secreting anti-human CD123 antibody.The antibody has strong valence with L929-CD123 cells.