T Cell Therapy In Humanized Mice With Human Immunity And Autologous Acute Lymphoblastic Leukemia

Background and objective:Acute lymphoblastic leukemia is clone proliferative disease of lymphocytic progenitor cells.Although B-ALL remission rate is gradually improved by using current treatment methods,the proportions of patients with drug resistance and relapse are high,while the rate of secondary remission is low.Anti-CD19 CAR T cell therapy for B-ALL can target and lyse leukemia cells,largely improve the remission rate of relapsed and refractory patients,which also gain opportunities for hematopoietic stem cell transplantation.However,due to the escape of leukemia cells from therapies and the existence of minimal residual disease in bone marrow.Because of the protective mechanism in tumor microenvironment(bone marrow niche),long-term remission hardly be achieved.Also,patients in the clinical trials of anti-CD19 CAR T cells showed a series of treatment-related adverse reactions,which would make the patients suffer a lot.Currently,there is still a lack of animal models to simulate B-ALL patients receiving homologous CAR T therapy.In addition,the treatment method to enhance the elimination of minimal residual disease by CAR T cells needs further researches.In this study,we established a humanized mouse(hu-mouse)model with a functional human immune system and genetically-matched(autologous)primary B-ALL,which permits to model CD19-targeted CAR T cell therapy in immune-competent hosts without allogeneic or xenogeneic immune responses.B-ALL cells express CXCR4,which is related to homing,treatment resistance and disease relapse of leukemia cells.By blocking SDF-1 /CXCR4 pathway,explore the method of enhancing T cells’ immunity to lyses B-ALL cells.Methods:Part 1,Established the model of anti-CD19 CAR T cell therapy in humanizedmice with human immunity and autologous leukemia.1.The human B-ALL humanized mouse model was established.Meanwhile,human T-cells were isolated from humanized mice,and anti-CD19 CAR T cells were prepared.B-ALL humanized mice were treated with homogenic anti-CD19 CAR T cells.2.Analyzed the leukemia cells,anti-CD19 CAR T cells and other immune cells in the mouse model,and detected the proliferation and survival of anti-CD19 CAR T cells in mice,proliferation and killing of leukemia cells by FACS.3.Checked the serum cytokines and survival of mice,and observed the correlation between the release of cytokines,the anti-leukemic effect after and the therapeutic effect of CAR T cells.Part2,Harnessed the potency of donor leukocyte infusion to eradicate leukemia by blockade of CXCR4.1.Established B-ALL NSG mice and allogeneic T cells infusion to lyses leukemia cells in vivo.2.Mobilized Leukemia cells into peripheral blood by CXCR4 antagonist AMD3100.3.Tested the proportion and count of various cells in peripheral blood of mice to evaluate GVL effect and GVHD.4.Cheked the survival of mice,detected the number and proportion of leukemia cells in the organs of mice.5.Adoptive transfered bone marrow cells of treated mice to secondary NSG mice to monitor the long-term remission of the disease.Results:Part 1,Established the model of anti-CD19 CAR T cell therapy in humanized mice with human immunity and autologous leukemia.1.Humanized mice were established through the transplantation of human embryonic thymus and fetal liver CD34+ cells.The human cell chimerism rates of humanized mice were over 49% in week 7 after transplantation.Established B-All humanized mouse model.2.The T-cells in the spleen of humanized mice were isolated.Prepare anti-CD19 CAR T cells and the control CAR T cells.The CAR T cell transfection rates were73% and 45.3%,respectively.3.B-ALL humanized mice received CAR T cell infusion.At week 1,2 and 4after the infusion of CAR T cells,B lymphocytes were significantly less in the CD19 CAR T cell treatment group than in the control CAR T cell treatment group,p values were 0.0144,0.0047 and 0.0059 respectively.At week 6 and 8,the leukemia cells in the CD19 CAR T cell treatment group were significantly less than those in the control CAR T cell treatment group,p values were <0.0001 and 0.0048 respectively.4.Cytokines in serum of B-ALL humanized mice after the treatment of CD19 CAR T cells.At week 1 after treatment,TNF-α and IL-10 in the CD19 CAR T group were significantly higher than those in the control CAR T group,p values were 0.0095 and 0.0422 respectively.At week 4 after the treatment,INF-γ level in the CD19 CAR T group was significantly higher than control CAR T group,p value was 0.0161.5.CD19 CAR T cell therapy extended the survival time of B-ALL humanized mice.The survival time of mice in the control CAR T cell therapy group were 51~82days(median survival time was 72 days),and the survival time of mice in the CD19 CAR T treatment group were 86~>120 days(median survival time was 99 days)after the CAR T cell therapy.The survival time of CD19 CAR T cell group was significantly longer than that of mice in the control CAR T group(p value was 0.003).Part2,Harnessed the potency of donor leukocyte infusion to eradicate leukemia by blockade of CXCR4.1.B-ALL NSG mice received allogeneic DLI treatment.The proportion of leukemia cells in bone marrow of mice was significantly higher than in peripheral blood,spleen,liver,lung and kidney(P<0.001).2.Leukemia cells of B-ALL NSG mouse expressed CXCR4.3.B-ALL NSG mice were injected with AMD3100 or PBS to mobilize leukemia cells to peripheral blood or as control.The proportion and count of peripheral blood cells in AMD group were significantly lower than those in PBS group on the 7th day after the first dose,p values were 0.0359 and <0.001 respectively.While there was no significant difference in peripheral blood leukemia cells in AMD group on the 21 st and 28 th days.There was no statistically significant difference in leukemia cells in mice organs.4.CXCR4 inhibitor enhances DLI GVL effect.Treat the B-ALL mice with allo-DIL with AMD3100/PBS.The level of peripheral blood leukemia cells in the DLI/AMD treatment group was significantly lower than that in the DLI /PBS control group at day 15,the p value was <0.0001.On day 25 after DLI,the proportion of bone marrow leukemia cells in the DLI/AMD group was significantly lower than that in the DLI / PBS group,p value was 0.0075.5.CXCR4 inhibitor combined with DLI can also affectively lyses leukemia in the more aggressive B-ALL NSG model.Two rounds of DLI combined with AMD/PBS were given to B-ALL NSG mice with rapid proliferation of leukemia cells.On day 31 after the first DLI,the percentage and count of peripheral blood leukemia cells in AMD group(2/5 mice without GFP+ cells detected)was significantly lower than that in PBS group,P values were 0.0098 and 0.007 respectively.There were significant differences in mouse bone marrow leukemia cell counts,p value was0.0174.DLI combined with AMD/PBS mouse bone marrow as first-generation mice,and bone marrow was adaptive transplanted to NSG mice(as 2nd-generation).Peripheral blood leukemia cells in the 2nd generations of mice in the AMD group were significantly lower than 2 generations PBS group.P values in the 5,6,7 and 8 weeks were 0.033,0.0077,0.0133 and 0.0829 respectively.1 2nd-generation mouse of the AMD group leukemia cells were not detectable for 23 weeks.Conclusions:1.CD19 CAR T cells constructed by human mouse spleen T lymphocytes can proliferate in vitro.2.CD19 CAR T cells constructed by spleen T cells of humanized mice can lyse common human B lymphocytes as well as leukemia cells in B-ALL humanized mice.3.Cytokines increase in the serum of B-ALL humanized mice post ant-CD19 CAR T cells treatment.4.The survival and expansion of CD19 CAR T cells are positively correlated with the treatment results.5.The B-ALL human mouse model of CD19 CAR T cell therapy can simulate the clinic,and provided a model for evaluating the anti-tumor effect of CAR T cells and improving the treatment.6.B-ALL cell in the bone marrow is less sensitive to T cell killing than peripheral organs.7.CXCR4 inhibitor AMD3100 can effectively mobilize leukemia cells in the bone marrow into the peripheral organs.8.In B-ALL NSG mouse model,AMD3100 alone can not effectively reduce leukemia cells.9.AMD3100 mobilization does not increase the GVHD effect of allogeneic DLI.10.CXCR4 inhibitor enhances the anti-leukemia effect of allogeneic T cells.