| ObjectiveClinical study,using CD19-CAR-T cells to treat relapsed(refractory)B-ALL with CD19,to observe the efficacy and adverse reactions.In vitro study.it’s to observe the affect of the optimum proportion of CD19-CAR-T cells and tumor cells and drugs including GCS and matrine on the anti leukemia effect of CD19-CAR-T cells.To analyze synthetically,as to provide a theoretical basis for effective treatment and even cure B-ALL with CD19-CAR-T cells.Clinical study1. Material and methodsSelecting relapsed(refractory)CD19+-B-ALL patients, to separate PBMC and CD3+-T cells,to use lentiviral vector with CD19 and CD137 to infect the T cells,gaining the 2th generation of chimeric CD19 receptor T cells(CD19-CAR-T cells). To detect transfection efficiency and lymphocyte subsets by flow cytometry,and killing effects by LDH assay.And to use pretreatments according to the tumor load before infusing,to monitor closely the condition changes and deal with adverse reactions,and to evaluate the therapeutic effect after one week,and to follow and treat regularly.2. ResultsAfter one week,to evaluate the therapeutic effect on 12 B-ALL patients who receive the CART therapy for the first time.The result is 75%(9/12) with tumor cell cleared,16.67%(2/12) without tumor cell cleared, 8.33%(1/12) died before evaluating. Early adverse reactions are ardent fever, increased heart rate, fall of blood pressure, shortness of breath, nausea, vomiting, edema, the function damage of liver and kidney, higher IL-6levels.100%(12/12) have different degrees of CRS. The most ones can be controlled,only 8.33%(1/12) died of severe adverse reactions. Lately,all have mild low gamma globulin serum,but don’t use gamma globulin for replacement therapy.In vitro study1. Material and methodsTo mix culture CD19-CAR-T cells and CD19+-K562 with different effect target ratios, to observe the killing effect of CD19-CAR-T cells on CD19+-K562 and the release of IL-6 by CCK 8 and ELISA respectively, finally,to select an appropriate effect target ratios to continue experiment.To use different concentrations of Matrine on co-cultured CD19-CAR-T cells and CD19+-K562, to observe the effect of drugs on the killing effect and the release of IL-6 by CCK 8 and ELISA; using different concentrations of GCS on CD19-CAR-T cells, to observe the effect of drugs on the proliferation of CD19-CAR-T cells and the killing effect by CCK 8.As to observe the effects of drugs on the anti leukemia effect of CD19-CAR-T cells.2. Results2.1 different effect target ratios:the killing effect of CD19-CAR-T cells on CD19+-K562To mix culture CD19-CAR-T cells and CD19+-K562 with different effect target ratios(2.5:1,5:1,10:1,20:1),then to detect the killing effect of CD19-CAR-T cells on CD19+-K562 and the release of IL-6 by CCK 8 and ELISA respectively. Results show that as the target ratio increasing,the killing effect of CD19-CAR-T cells on CD19+-K562 is enhanced, but the release of cytokines is also increased(P < 0.05).So comprehensively selecting the effect target ratio of 10:1 as the appropriate effect target ratios.2.2 The morphologic variations of CD19-CAR-T cells and CD19+-K562To mix culture CD19-CAR-T cells and CD19+-K562 for 0h and 36 h by 10:1, to observe cells stained by Wright-Giemsa with microscope. Results show that after co-culturing,the number of cells reduces evidently, the cell and nuclear volume of CD19+-K562 shrink,and two kinds of cells’ morphology are incomplete.2.3 The effect of matrine on the killing effect of CD19-CAR-T cells on CD19+-K562To mix culture CD19-CAR-T cells and CD19+-K562 by 10:1, then use matrine at different concentrations(0 mg/ml, 0.5 mg/ml, 1.0 mg/ml, 2.0 mg/ml) to treat co-cultured cells for 24 h and 36 h, to detect the killing effect of CD19-CAR-T cells on CD19+-K562 and the release of IL-6 level respectively. Results show that comparing all the drugtreatment groups with the control group, the killing effects are increased(P < 0.05),but IL-6 levels don’t increase evidently(P > 0.05);and between the drug treatment groups, the killing effect and IL-6 levels have no obvious difference(P > 0.05).2.4 The effect of GCS on the killing effect of CD19-CAR-T cells on CD19+-K5622.4.1 The effect of GCS on the proliferation of CD19-CAR-T cellsUsing GCS at different concentration(0 mg/ l, 10 mg/l, 20 mg/ l,40 mg/l) to treat CD19-CAR-T cells for 24 h, then to detect the inhibition effect of drug on cells by CCK 8.Results show that GCS can inhibit the proliferation of CD19-CAR-T cells, and the inhibition rates of different drug treatment groups have obvious difference(P < 0.05).2.4.2 The effect of GCS on the killing effect of CD19-CAR-T cells on CD19+-K562Using GCS at different concentration as the above to treat CD19-CAR-T cells for24 h, the to mix with CD19+-K562 respectively by 10:1, finally,to detect the killing effect by CCK 8. Results show that comparing all the drug treatment groups with the control group, the killing effect decreased(P < 0.05); but between the drug groups,the killing effects have no obvious difference(P > 0.05).ConclusionThe reaction rate of the treatment of relapsed(refractory)B-ALL with CD19-CAR-T cells is high,but recurrence may occur. Early after infusing,CRS is 100%,but most adverse reactions can be controlled,a few may lead death.Lately,all CR ones have mile hypogammaglobulinemia,but need not to treat.As the target ratio increasing,the killing effects and the release of cytokines both are increased. To analyze synthetically, CART therapy is a prospective treatment for ALL,and the reasonable application of aided drug maybe can enhance the killing effect and control the degree of adverse reactions,but the return transmission scheme and the treatment after CR is needed to continue to explore. |
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