| Part one:Clinical study of chimeric antigen receptor modified T cells for the treatment of relapsed and/or refractory acute lymphoblastic leukemia in children and adolescentsBackground:Acute lymphoblastic leukemia(ALL)accounts for the vast majority of the acute leukemia in children,accounting for about 65%to 70%of the cases.With the treatment according to the degree of risk,and the complete remission rate can reach more than 95%.The 5 years event-free survival can reach 80%～90%,but there are 10%～20%of children would still relapse or refractory to chemotherapy after traditional chemotherapy or hematopoietic stem cell transplantation,which account for an important cause of leukaemia related deaths.In particular,patients with relapsed disease after allogeneic hematopoietic stem cell transplantation(allo-HSCT)have a worse prognosis.In recent years,chimeric antigen receptor modified T cells(CAR T)technology has been widely used in r/r B-ALL.However,the experience in pediatric patients is limited,and the study of the side effects and long-term outcomes in children is still insufficient.Objective:To investigate the short-term and long-term outcomes of CD19-CAR-T in the treatment of r/r B-ALL in children and adolescents,and to analyze the influencing factors to the consequence,in order to provide some idea for early intervention and prognostic indicators,and to improve the outcome of refractory recurrent acute lymphoblastic leukemia.Methods:1.In this study,By consulting the electronic medical record information on the hospital information system,The inpatients and outpatients were observed and recorded,and the discharged patients were also followed up by telephone.The Children and adolescents patients with r/r B-ALL aged ≤20 years who has underwent CAR-T therapy from August 2016 to June 2020 were enrolled in,whose preliminary data were collected.After receiving CAR-T therapy,We recorded the number of CAR-T cells infused,and the adverse reactions and therapeutic effects were recorded.Meanwhile,complications and long-term survival during infusion were followed up.2.SPSS 26.0 software was used to analyze the early effect and its related factors after treatment by Chi-square test.Kaplan-meier survival analysis,Cox risk analysis and Logrank were used to analyze The overall survival time(OS)and relapse-free survival time(RFS),as well as significantly related risk factors,were evaluated by means of testing,KW test was used to analyze the occurrence of adverse reactions and the relationship between the related factors,and patients were further divided into severe CRS group and non-severe CRS group according to the grade of CRS.The prediction model of severe CRS was established by ROC curve.P<0.05 indicated statistically significant difference,and P value was two-sided test.Results:1.75 children were included in this study,and the median follow-up time was 800.00(60 to 1452)days.The average number of transplanted CAR-T cells was 5.2×106(5× 106 to 1 ×107)/kg body weight,and the positive rate of CAR cells ranged from 17.13%to 74.38%.2.69.3%of patients developed cytokine release storm(CRS)within 1 week after CAR-T infusion.It was found that patients with high disease burden before CAR-T infusion and high cytokine peak within 1 week after CAR-T infusion had higher SEVERITY of CRS.The number of CAR-T cells injected had no significant effect on the severity of CRS.In this study,ROC curves of the above factors for the diagnosis of severe CRS were respectively study,we found that in the inflammatory cytokines of serum ferritin(SF),procalcitonin(PCT),interleukin-6(IL-6),interleukin-10(IL-10),interferon-γ(IFN-γ),The area under ROC curve(AUC)was above 0.8,and the diagnosis accuracy of severe CRS may significant.3.There are 66 of the 75 patients who achieved complete remission(The overall response rate was 88.00%)after CAR-T infusion.It was found that the early remission rate was higher in the group which lymphoblastic cells<5%,while the time of relapse,the number of relapse,the number of CAR T cells transfused,gender,age and genetic factors of high-risk molecules had no significant influence on the early remission rate.4.Long-term follow-up find out that The median RFS time of the 75 children enrolled was 8.67months,and the median OS time was 13.33months.The following factors such as whether bone marrow reached M1 before CAR-T therapy,recurrence≥3 times,and whether HSCT was given after CAR-T affected the outcome of RFS and OS significantly.High-risk molecular genetic factors are adverse factors affecting OS,and the patients who relapsed after the former HSCT can still get better RFS after CAR-T.Multivariate analysis showed that≥3 times of pre-CAR recurrence was an independent risk factors for OS,and weather bridging HSCT followed CAR-T therapy was an independent risk factor for RFS and OS.Conclusion:CD19-CAR-T cells have a high complete remission rate for r/r B-ALL in children and adolescents,which is safe and effective.The lymphoblastic load before CART and the increased peak values of serum cytokines in the early period post-CAR-T correlated with the increased risk of severe CRS.The pre-CAR-T leukemia load was significantly associated with the early treatment response rate.The lymphoblastic load before CAR-T infusion,relapse of more than 3 times before CAR-T,and not bridging transplantation after CAR-T therapy,patients accompanied with high-risk genetic risk factors are adverse factors affecting overall survival,and patients who relapsed after transplantation can still benefit from CAR-T therapy.Part two:The experimental study of CD4CAR-T cell for the treatment of CD4positive malignant T cell hematological malignanciesT cell acute lymphocytic leukemia/lymphoma is a part of hematological malignancy in children and adolescents.The long-term treatment efficacy of T cell malignancy is not satisfactory.There is no very effecitve treatment options for r/r T cell malignancy,and the CAR-T treatment for T cell malignancy is not mature.Therefore,we studied CD4CAR-T immunotherapy in the treatment of T cell malignancy,in order to provide some supplements to the current CAR-T treatment research of T cell malignancy.The key to CAR-T treatment is to find the right CAR-T target.CD4 antigen is specifically expressed on the surface of CD4+T helper cells and parts of T-lymphoblastic leukemia/lymphoma,but not on the surface of normal hematopoietic stem cells.Therefore,CD4 antigen is one of research hot spot in the treatment of Tlymphoblastic leukemia/lymphoma by CAR-T.In this study,lentivirus was used as the plasmid vector,mouse anti-human CD4 monoclonal antibody was used as the single chain variable region sequence of extracellular monoclonal antibody,4-1BB was used as the costimulatory molecule,and CD3ζ chain was used as the intracellular signal domain to construct chimeric antigen receptor.CD4CAR-T cells were cultured to observe the expression efficiency of CD4CAR-T cells.Then the cultured CD4CAR-T cells were used to kill CD4-positive KARPAS 299 cells,and the secretion of cytokines and their targeted killing effect were observed,to investigate the effect of CAR-T cells against T cell leukemia in vitro and in vivo.Objectives:To explores the construction of anti CD4 chimeric antigen receptormodified(CD4CAR-T)and its killing function in vitro and in vivo,and study the of feasibility CD4CAR-T to treat the CD4-positive T lymphocytic leukemia.Methods:The single chain variable region fragment of CD4 monoclonal antibody,CD 137 co-stimulatory molecule and CD3ζ chain sequence were identified and integrated into a whole sequence,namely CD4CAR-T,using genetic engineering methods.DNA sequencing verified the correctness.The CD4-CAR fragment was then cloned into the lentiviral vector.Then,293T cells were co-cultured with shuttle plasmid,packaging plasmid pGag/Pol,pReV,pVSV-G)and CD4-CAR plasmid and transfected into lentivirus.The sequence of lentivirus was tested to see if it was correct.Activated T cells were infected with lentivirus,and CD4CAR-T cells were cultured in serum-free medium.Flow cytometry was used to detect the transfection rate,the growth and expression of CD4CAR-T cells,and the expression of cytokines.CD4CAR-T cells and T-cell leukemia cells were co-cultured to detect the cytotoxic activity of CD4CAR-T cells in vitro.KARPAS 299-luc leukemia NSG mice were constructed to detect the killing effect of CD4CAR-T cells in vivo.Results:DNA sequencing showed that the gene sequence of lentivirus was correctly constructed,and CD4CAR-T cells were successfully prepared.DNA sequencing verified the correctness.The transfection rate was about 73.6%examined by flow cytometry after transfection of T cells with lentivirus.CD4CAR-T cells showed strong killing effect of CD4 positive leukemia,after co-culture of CD4CAR-T cells and KARPAS 299-luc cells.The NSG mouse model of KARPAS 299-luc T cell leukemia was successfully constructed,and the therapeutic effect of CD4CAR-T cells on leukemia in NSG mice was observed.The survival time of the treated mice was prolonged.Conclusion:We construct CAR-T cells targeted CD4 and demonstrate the anti-tumor effect on CD4+T leukemia cells through a series of experiments.In vitro experiment confirmed CD4CAR-T specific cytotoxicity to malignant CD4+T cell cells,and also demonstrated through in vivo experiment that CD4CAR-T cells can significantly inhibit leukemia progress in NSG mice model of KARPAS 299-luc cells and extend their survival.It provides an expansion of treatment ideas for the treatment of T lymphoblastic leukemia/lymphoma. |
