Analysis Of The Effect Of CAR-T Cells In Treatment Of Recurrent Refractory DLBCL

BackgroundsFor years,the basic treatments of cancer have included surgery,chemotherapy and radiotherapy,in recent years,we have the targeted drug therapy for cancer stem cells as well.Although these methods have helped to improve the treatment outcomes,the prognosis of most malignancies still remains poor.With the development of bioengineering techniques,researchers have developed chimeric antigen receptor(CAR)T cells which can kill tumor cells through cellμlar immunotherapy.As a new mode therapy for malignant tumor,CAR-T cells can recognize and combine the corresponding antigen on tumor cell surface,further stimμlate T cells secreting cytokines,thus producing the killing effect of tumor cells.At present,the application of CD19 CAR-T cells in the treatment of hematology malignances has made a breakthrough.The total remission rate(CR)for acute B-lymphocytic leukemia(B-ALL)can reach 70%-94%,which greatly improving the patient’s living condition,and the clinical resμlts of CD19 CAR-T cell therapy for B cell lymphoma showed a 35%-52% remission rate.However,recurrence of the disease was still observed in clinical trials for both B-ALL and B-cell lymphoma.Many literatures have also reported possible factors leading to recurrence,such as immune escape due to negative CD19 antigen on tumor cells.So,it is hoped that the combined action of mμltiple target antigens can reduce the immune escape.Diffuse large B cell lymphoma(DLBCL)is the most common malignancy of the blood system.The current treatment options for DLBCL include chemotherapy,radiotherapy and chemotherapy combined with targeted drugs such as Rituximab.Although the use of targeted drugs can increase the response rate of disease,recurrence of lymphoma is still unavoidable.The resμlts of the treatment of DLBCL by CD19 CAR-T cell showed that was effective.However,its curative effect is obviously lower than that of B-ALL,and it is also impossible to avoid recurrence.In the treatment of CAR-T cells,IFN-γcytokines kill tumor cells by activating T cell function.It also stimμlates monocytes to secrete inflammatory cytokines such as TNF-alpha,IL-6 and IL-10,which regμlate immune responses.Mμltiple studies have shown that malignant lymphoma cells can secrete mμltiple cytokines as well,The resμlts showed that IL-10 increased in malignant lymphoma,and the content of IL-10 was correlated with the prognosis and efficacy of lymphoma.We have to clear that the IL-10 cytokines detected after the treatment of B-cell lymphoma by CAR-T cells are caused by T-cell-mediated secretion or lymphoma cells.we need further analysis that if they related to relapse of DLBCL.ObjectiveThis study is divided into two parts.Part 1,To prepare CD19 CAR-T cells by transfection of CD19 gene to human peripheral blood T lymphocytes.To evaluate the quality of cells and to test the cytotoxicity of cells in vitro.Part 2,Using CD19 CAR-T cells treat patients with recurrent refractory DLBCL,observe the curative effect and analyze the level change of IL-10 in patients after treatment,and analyze the correlation between the level change of IL-10 and the recurrence of DLBCL.MethodsPart 1 The CD19 CAR gene was prepared,and the peripheral blood T lymphocyte of DLBCL patients was treated with recurrent infection.Observe the morphology of the cell,detecte the transfection and evaluate the in vitro killing effect.Part 2 The treatment of 11 patients with recurrent refractory DLBCL with CD19 CAR-T cells was evaluated.analyze the correlation between the level change of IL-10 and the recurrence of DLBCL.ResultsThe CD19 CAR-T cells were well-formed and well-developed,The resμlt of flow cytometry indicated that CD19 CAR-T cells met the needs of treatment and experiment.The killing ability was significantly better than that of 293 T cells without CAR structure(P<0.05).11 patients with recurrent refractory DLBCL treated with CD19 CAR-T cells.The CR rate was 50 % and the ORR rate was 60 %,There was no adverse reaction due to transfusion of CAR-T cells.The change trend of CAR gene copy number in peripheral blood was similar to that of cytokine content.The effect of CD19 CAR-T cells has no correlation with the dose of retransmission(P=0.08),It has nothing to do with whether there was a bone marrow invasion(P=0.487).2 Patients recurred around 3 months after treatment,Reverse increase in the level of cytokine IL-10 when the CAR gene level decreased in peripheral blood,There was a statistically significant difference between the levels of IL 10 in relapse patients and those without relapse(P=0.009).Conclusion1.CD19 CAR-T cell quality is in accordance with the experimental requirements,and in vitro killing ability is strong.2.CD19 CAR-T cells were effective in treating patients with recurrent refractory DLBCL.The CR rate was 50 % and the ORR rate was 60 %.there was no adverse events during treatment.3.The efficacy of CD19 CAR-T cells has no correlation with the dose of redelivery and had no relationship with bone marrow invasion.4.Increased level of IL-10 in peripheral blood of patients with recurrent disease,correlated with recurrence.