Study On Generation Of Cord Blood-derived CD19-CAR T Cells And The Anti-tumor Effects In Diffuse Large B-cell Lymphoma

【Background】 Chimeric antigen receptor T cell(CAR T)therapy is one of the most significant breakthroughs in the field of hematologic malignancies.Currently,autologous CAR T cell therapy has been approved by the US Food and Drug Administration as a second-or third-line treatment for large B-cell lymphoma.However,most patients experience lymphocyte deficiency and functional impairment after undergoing multiple lines of chemotherapy.Moreover,the production of autologous CAR T cells is personalized and time-consuming,and some patients may experience disease progression or treatment-related complications while waiting for autologous CAR T cell infusion,thereby missing the opportunity for CAR T cell therapy.Lastly,the individualized manufacturing process for autologous CAR T cells leads to prohibitively high costs,making it difficult to afford.These factors not only affect the efficacy of autologous CAR T cell therapy but also hinder its widespread application.The use of "off-the-shelf" allogeneic CAR T cell therapy can effectively address the aforementioned issues.However,the transplantation of allogeneic donor T cells may lead to life-threatening graft-versus-host disease(GVHD)and can be easily recognized and eliminated by the host immune system.Currently,universal CAR T cells mainly come from peripheral blood mononuclear cells(PBMCs)of healthy donors,with a small number of cells derived from cord blood(CB).Previous studies have demonstrated that allogeneic CB transplantation exhibits effective anti-tumor activity with lower relapse rates and GVHD incidence.Activated CD8+ CB T cells,without antigen stimulation,exhibit stronger proliferative capacity compared to T cells derived from peripheral blood(PB).After antigen stimulation,these immature CB T cells can demonstrate cytotoxic effects.Additionally,CB as a source is more stable.Therefore,we believe that CB-derived T cells may serve as an effective source for CAR T cells.Exploring CB as a viable source for universal CAR.Purpose: Autologous chimeric antigen receptor(CAR)T cell therapy is one of the most significant breakthroughs in hematological malignancies.However,a three-week manufacturing cycle and ineffective T cell dysfunction in some patients hinder the widespread application of auto-CAR T cell therapy.Studies suggest that cord blood(CB),with its unique biological properties,could be an optimal source for CAR T cells,providing a product with ’off-the-shelf’ availability.Therefore,exploring the potential of CB as an immunotherapeutic agent is essential for understanding and promoting the further use of CAR T-cell therapy.Experimental Design:1.Construction of CB CD19-CAR T cells(1)Based on the previously reported sequence information,construct the CD19-CAR gene(CD19 sc Fv-CD8-4-1BB-CD3)and clone it into a lentiviral vector plasmid.(2)Extract T cells from CB and use a lentiviral transfection system to transfer the CD19-CAR gene into CB T cells,constructing second-generation CAR T cells targeting CD19.Use flow cytometry to detect the transfection efficiency of CD19-CAR T cells.(3)Use flow cytometry to detect the proportion and immunophenotype of CD4,CD8,TCR-α and TCR-γ T cells in CB T cells and CB CD19-CAR T cells.(4)Use RNA-seq to detect the differences in gene expression between CB T cells and CB CD19-CAR T cells.2.Antitumor effects of CB CD19-CAR T cells on DLBCL cells in an in vitro(1)Use CFSE to label target tumor cells.(2)Co-culture CB CD19-CAR T cells with CFSE-labeled ALL cell lines BV173 and K562 at 1: 1,2: 1,5: 1,and 10: 1 effector-to-target ratios for 24 hours.Use flow cytometry to detect the percentage of FVS-positive tumor cells.(3)Co-culture CB CD19-CAR T cells with CFSE-labeled DLBCL cell lines SUDHL-4 and DB at 1: 1,2: 1,5: 1,and 10: 1 effector-to-target ratios for 24 hours.Use flow cytometry to detect the percentage of FVS-positive tumor cells.(4)Co-culture CB CD19-CAR T cells with BV173 and SUDHL-4 cells at 1: 1effector-to-target ratio for 48 hours.Use flow cytometry CBA multiplex assay to detect the cytokine secretion levels of CB CD19-CAR T cells in the co-culture supernatant.3.Antitumor effect of CB CD19-CAR T cells in DLBCL xenograft model(1)1x106 SUDHL-4 cells were injected into the dorsal side of BALB/c nude mice by subcutaneous injection.After tumor formation,randomly divide the mice into three groups and inject 1x106 CB CD19-CAR T cells,CB T cells(normal control,T cells not transfected with CD19-CAR lentivirus),or an equal volume of PBS via tail vein.(2)Weigh the mice every 2-3 days,measure the size of the tumor,and record tumor progression.Terminate the experiment when mice show signs of imminent death.Observe GVHD-related symptoms in mice.4.Immunophenotype and gene changes of CB CD19-CAR T cells(1)Co-culture CB CD19-CAR T cells with SUDHL-4 cells at 1: 1 effector-to-target ratio for 48 hours.Use flow cytometry to detect changes in the immunophenotype of CB CD19-CAR T cells,as well as the expression levels of immune checkpoints associated protien TIM-3 and PD-1.(2)Co-culture CB CD19-CAR T cells with SUDHL-4 cells at 1: 1 effector-to-target ratio for 48 hours.Use CD3-positive selection magnetic beads to sort out the co-cultured CB CD19-CAR T cells and the CB CD19-CAR T cells that were not co-cultured for RNA-seq analysis.Results:1.In this study,CD19-CAR lentiviral vectors were successfully used to transfect CB T cells.Flow cytometry analysis showed a transfection efficiency of 60.8%.Flow cytometry analysis revealed that CB CD19-CAR T cells and CB T cells exhibited consistent culture conditions,and no significant differences in proliferation were observed after cultivation.Transfected CB CD19-CAR T cells did not show significant differences compared to CB T cells in terms of immune phenotypes,including CD4,CD8,TCR-α,and TCR-γ.Additionally,RNA-seq results indicated that CB CD19-CAR T cells displayed gene changes related to membrane transport,endocytosis,and immune response,potentially caused by the assembly of CARs and enhancing membrane functionality and T cell immune response.2.In vitro experimental results using CB CD19-CAR T cells targeting CD19+ DLBCL cells demonstrated the effective killing of CD19+ ALL cell line BV173 and DLBCL cell line SUDHL-4 compared to CB T cells.Flow cytometry CBA multi-factor analysis showed that CB CD19-CAR T cells secreted higher levels of cytokines,including IL-2,IL-4,IL-6,IL-10,TNF-α,and IFN-γ,when co-cultured with BV173 and SUDHL-4 cells,compared to CB T cells.CB CD19-CAR T cells exhibited specific targeting and killing of BV173 and SUDHL-4 cells,accompanied by significant cytokine secretion.However,the cytotoxicity of CB CD19-CAR T cells against SUDHL-4 cells was not dependent on the effector-to-target ratio.3.In vivo experiments using CB CD19-CAR T cells targeting DLBCL xenograft mouse models demonstrated effective inhibition of DLBCL tumor growth,while the tumor growth in the PBS group and CB T cell group was rapid,resulting in higher tumor burden compared to the CB CD19-CAR T cell intervention group.Safety evaluation and GVHD scoring indicated no observed cytotoxicity or GVHD symptoms during CB CD19-CAR T cell therapy.4.After co-culturing with tumor cells,CB CD19-CAR T cells showed a loss of the CCR-7phenotype,transitioning from a naive T cell phenotype to a terminal effector memory T cell(TEMRA)phenotype.Following co-culture with tumor cells,CB CD19-CAR T cells exhibited significantly increased expression of the immune checkpoint protein TIM-3,while PD-1expression showed no significant differences.RNA-seq results revealed changes in genes associated with T cell exhaustion,co-stimulatory molecules,and memory cells in CB CD19-CAR T cells after co-culture,possibly contributing to the development of exhaustion following the anti-tumor effect of CB CD19-CAR T cells.Conclusion:The research findings of this study indicate that:(1)Our study successfully constructed a CD19-CAR that efficiently transfects CB T cells.Compared with CB T cells,CB CD19-CAR T cells did not exhibit any changes in immune phenotype,but RNA-seq revealed changes in genes related to membrane transport,endocytosis,and immune responses.(2)CB CD19-CAR T cells effectively killed ALL cell lines and DLBCL cell lines,while being activated by target cells and secreting large amounts of cytokines such as IL-2,IL-4,IL-6,IL-10,TNF-α,and IFN-γ.In particular,the killing effect of CB CD19-CAR T cells on the DLBCL cell line SUDHL-4 was not dependent on the effector-to-target ratio.(3)CB CD19-CAR T cells effectively inhibited DLBCL tumor growth in mice.Safety evaluation and GVHD scoring showed no observed cytotoxicity or symptoms of GVHD with CB CD19-CAR T cell therapy.(4)After co-culture with tumor cells,CB CD19-CAR T cells showed loss of CCR-7 phenotype,transformed from a naive T cell phenotype to a TEMRA phenotype,and showed a significant increase in TIM-3 expression.At the same time,RNA-seq revealed changes in T cell exhaustion-related genes,co-stimulatory genes,and memory cell-related genes in CB CD19-CAR T cells after co-culture.Therefore,we believe that CB is an effective source of universal CAR T cells,and CB CD19-CAR T cells are a promising therapeutic strategy for DLBCL.The unique biological characteristics and high utility of CB T cells make CB CAR T cell therapy feasible.