| B-cell leukemia and lymphoma are the most common subtypes of hematological malignancies.Relapse and refractory are the main cause of treatment failure and nearly one-third of patients died as a result.In the past five years,the treatment by chimeric antigen receptor specific T cells(CART)that specifically recognize CD19 on the surface of B cells has achieved remarkable results in clinical trials of relapsed or refractory B-cell lymphoma,which raises a worldwide corporation of the development and research of CART cells.The CAR structure is mainly composed of three parts:the extracellular antigen binding region,the transmembrane region and the intracellular signaling region of the T cell receptor.At present,preclinical and clinical trials of most CD19-CART-cells at home and abroad use second-generation CART technology containing CD28 or 4-1BB co-stimulatory molecules.To date,there have been more than 80 publicly registered clinical trials of B-cell lymphoma/leukemia globally.However,the effect of clinical treatment of anti-CD 19-CART cells performed in China is far behind the international level.The main reason is that the CAR gene structure is less ideal,the in vitro gene modification technology is unstable and the preclinical CD 19-CART-cell immunotherapy efficacy and safety study data are not sufficient.The preparation efficacy and safety of CD19-CART-cells products is particularly important for regulating CART cell therapy.Therefore,the CD 19-CART preclinical cell preparation process is under standardized and the quality control is under improved.In the CART-cells immunotherapy,the CART cells bind directly to the tumor antigen and initiate non-MHC-restricted cellular immune killing.Its advantages lie in the efficient and specific binding of tumor antigens which increases the effectiveness,specific targeting and persistence of T cell killing.Meanwhile,study of Tomonori Tsukahara et al.indicates whether CART can rapidly return to the tumor site and maintain a high concentration is related to the outcomes of CART immunotherapy.The number and distribution of CART cells determine whether they can bind to tumor antigens and clear them in vivo.Therefore,to observe and explore the distribution,homing and amplification of CART cells during immunotherapy can help improve the outcome of CART cell immunotherapy.The current worldwide phase I clinical trial on CD 19-CART cells immunotherapy has achieved significant progress in the treatment of relapsed or refractory B-cell acute leukemia,chronic leukemia,and lymphoma.However,some patients still relapsed after achieving remission or remission after CD19-CART-cells immunotherapy.Therefore,optimizing CART treatment is particularly important in clinical practice.At present,most of the clinical trials on CD 19-CART at home and abroad do not sort T cells.Therefore,the subtypes of CD 19-CART infused in each patient in the clinical trial were different.This difference of subtypes will result in differences in the efficacy of CD 19-CART treatment among different patients,making it difficult to achieve clinical standardization of CD 19-CART immunotherapy for each patient.Research by Daniel Sommermeyer et al.of the Fred Hutchinson Center for Cancer Research for the first time sorted CD 19-CART to study the functional differences between different subtypes of T cells,confirming that CD4+ and CD8+ T cells play different and interdependent roles role in anti-tumor activity of CD 19-CART.CD8+ T cells are mainly responsible for the killing and elimination of tumor cells and play a major role in anti-tumor immunity.CD4+ T cells are mainly responsible for secreting cytokines,promoting the proliferation of CD8+ T,and assisting CD8+ T cells to kill tumors.CD8+ T cells and CD4+T cells in the CD19-CART immunotherapy promote each other and perform interdependent effects.However the best ratio and effective dose of CD4+and CD8+T cells have not been identified in the study.Meanwhile,CD4 and CD8 were further divided into primary T cells(TN),effector T cells(TE),central memory T cells(TCM),and effector memory T cells(TEM)and their roles in CART immunotherapy were respectively explored.This method can hardly be achieved in CART clinical trials and clinical applications.Therefore,it is necessary to explore the optimal ratio and clinical effective dose of CD4+ and CD8+ T cells.Objectives:To establish a CD 19-CART expansion system in vitro,evaluate its safety and efficacy and explore the distribution,homing and amplification of CD 19-CART cells after transfusion in vivo.Meanwhile,to optimize CD19-CART immunotherapy and achieve clinical standardization and industrialization of CD 19-CART cell immunity,the role of CD4+and CD8+ T cells in CD 19-CART immunotherapy and the optimal combination ratio as well as the effective dose of CD4+and CD8+T cells were explored.Materials and Methods:(1)We constructed the CD 19-CAR expressing four-plasmid lentivirus vector,established the CD 19-CART cells based on amplification system in vitro and evaluated the efficacy and safety of CD19-CART-cells immunotherapy by cell lysis test in vitro and in vivo.(2)We constructed CD19-CART-Fluc cells with fluorescent label and observed the distribution,homing,and amplification of CD 19-CART cells in vivo.(3)PBMCs were sorted into CD4+and CD8+T cells by immune-magnetic positive sorting method.Then CD4+CD19-CART cells and CD8+ CD 19-CART cells were constructed by using lentivirus transfection method,respectively.They were then combined at different CD4/8 ratios(CD4,CD4/8 3:15 1:1,1:3)and CD4+and CD8+CD 19-CART cells were observed by in vitro and in vivo cell lysis test.And the results of cell lysis test were analyzed to choose a best ratio.(4)Based on the third part,it was found that CD8+CD19-CART cells were mainly responsible for the early killing and clearance of tumor cells,which was critical for CD 19-CART-cells immunotherapy.Therefore,three doses of CD8+CD 19-CART cells were chosen for following tests:low dose group(2 × 106),middle dose group(5 X 106),and high dose group(1 × 107).The lowest effective dose of CD8+CD 19-CART cells would be determined by in vivo cell lysis test.CD4+ CD 19-CART cells were mainly responsible for secreting cytokines,promoting tumor immune killing,assisting the killing by CD8+CD 19-CART cells and preventing tumor recurrence in a long period after CART-cells immunotherapy.After determining the effective dose of CD8+CD 19-CART cells,the CD4+CD 19-CART dose was determined by in vivo cell lysis test.Based on the observation of cell lysis test of different doses of CD4+CD 19-CART and fixed dose of CD8+CD 19-CART,the minimum effective dose of CD4 CD 19-CART was determined finally.Results:(1)CD 19-CART cells were successfully constructed and expanded in vitro.Tumor lysis tests in vitro and in vivo showed that the constructed CD 19-CART cells could effectively kill CD 19 tumor antigen-positive cells.The high dose group CD 19-CART cells owned better killing effect than the low dose group.(2)CD 19-CART could go home quickly after reinfusion in mouse with CD 19 antigen-positive lymphoma,bind to tumor antigen,survive in vivo for a long period and induce immune surveillance and immune memory.(3)The tumor lysis test in vitro with different CD4/8 ratios showed that CD8+CD 19-CART cells were mainly responsible for direct killing of tumor cells and CD4+CD 19-CART cells were mainly responsible for secreting cytokines of human IL-2,IL-10,IFN-y and TNF-a.Tumor cell lysis test in vivo showed that the groups of CD4/8 1:1 and 1:3 supported the longest survival.Early clearance of the tumor by CD8 CD 19-CART cells is the primary once its dose was sufficient,otherwise the tumor relapsed quickly.Meanwhile,CD4 CD 19-CART cells were responsible for assisting CD8 CD 19-CART cells in killing tumor cells.In the absence of CD4 CD19-CART cells,the tumor would relapse even if the tumor cells are completely removed by CD8 CD 19-CART cells.(4)The tumor lysis tests of different doses of CD8 CD 19-CART cells showed early eradication of tumor cells in the middle dose group(5×106)and high dose group(1×107),but not sufficient in the low dose group(2×106)with rapid relapse.Therefore,the effective dose of CD8+CD 19-CART cells was determined as 5×106.After fixing the effective dose of CD8 CD 19-CART cells,the lowest efficient dose of CD4+CD 19-CART cells was at least 1/3 of the effective dose of CD8 CD19-CART cells to ensure long-term remission after CART-cells immunotherapy with longer survival.Conclusions:(1)The use of 4-1BB co-stimulatory molecules for second-generation CAR design and the construction of CD 19-CART cells with four-plasmid lentiviral transfection can ensure the safety and efficacy of CD 19-CART cells immunotherapy which is consistent with clinical trials and preclinical trials worldwide.(2)The targeted homing and survival ability determined the quality of CD 19-CART,which is crucial for the result of tumor lysis in vivo and in vitro.(3)CART cells sortation can optimize clinical outcome of CD19-CART-cells immunotherapy,in accordance with the effective dose of CD4 CD 19-CART cells and CD8 CD 19-CART cells,which helps to avoid deviations in treatment effects due to different proportions of T-cell subsets among diferent patients and make the comparison of clinical outcomes among different centers easier.(4)CART cells immunotherapy will be standardized in the future.Therefore,the study of CART cells sorting of great importance,which requires further researches large scale clinical trials. |
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