Studies On The Abilities And Safety Of Anti-CD19 Chimeric Antigen Receptor T Cells In Killing Lymphomain Vivo

ObjectiveChimeric Antigen Receptor T Cell(CAR-T)therapy is an immunotherapy technology that uses genetic modification of T cells to achieve targeted tumor killing.At present,CAR-T cell therapy has achieved great results in the treatment of hematological tumors.However,the method of introducing CAR genes during the preparation of CAR-T cells is mainly transfected by virus.Whether the cost of preparation,pathogenicity or the potential for insertional mutagenesis during viral transfection,there are significant regulatory obstacles in clinical trials.The artificial antigen-presenting cells were independently constructed by our laboratory can be used as feeder cells to stimulate the proliferation of CAR-T cells in vitro,which not only improved the amplification efficiency,but also reduced the amount of exogenous cytokines added and reduced the cost of experiments.Therefore,it could reduce the financial burden of patients in clinical.In order to avoid the shortcomings of the virus transfection system,the CAR-T cells used in this experiment were prepared by the piggyBac transposon transformation system.Therefore,the purpose of this experiment was to verify the in vivo tumoricidal activity and safety of CAR-T cells produced under the new preparation system and stimulated by artificial antigen presenting cells(aAPC).As a preclinical study,it had been fully proved that CAR-T cells prepared under this culture system had significant in vivo tumoricidal activity,thereby promoting the treatment plan to enter the clinical stage as soon as possible.The main contents and experimental results of this study were as follows:MethodsAll experimentalmethods in mice were conducted in accordance with the approved guidelines specified by the AnimalExperimentation Ethics Committee of Yan'an Hospital Affiliated to KunmingMedical University.Section one:Establishment of the hematological tumor modelsTwo human Burkitt's lymphoma cells—NAMALWA and Raji which containing Luciferase and specifically expressing CD 19 antigen were used as tumor cells for the construction of the lymphoma mouse tumor-bearing models.SCID mice and NOD-SCID mice were used as mouse strains to construct human Burkitt's lymphoma mouse models.After the mouse model was constructed,the growth and distribution of tumor cells in mice were measured by a small animal in vivo fluorescence imaging system(IVIS).In order to confirm the successful establishment of the human Burkitt's lymphoma mouse models,after the tumors fully developed,the mice were sacrificed and the samples from peripheral blood mononuclear cell,bone marrow and spleen were detected by flow cytometry(FCM),as well as blood smear examination.Section two:Evaluation of the anti-tumor effects of CAR-T cells in vivoAfter successful establishment of the Burkitt's lymphoma mouse model,the lymphoma model mice were intravenously injected with anti-CD 19 chimeric antigen receptor T cells prepared by the new culture system,and the tumor develpment was detected by small animal in vivo fluorescence imaging system.The survival of mice was aslo record and analyzed,After experiment,the mice were sacrificed and the samples from heart,lung,liver,and brain were collected and fixed for further tested.The distribution of tumor cells in mice in the CAR-T cell group and in the saline-injected group were tested to verify the anti-tumor activity of CAR-T cells in mice.Section three:Validation of the safety of CAR-T cells and aAPC cells in vivoThe body weight were measured during the whole experiment.To determine whether the CAR-T treatments had adverse reactions to mice,the pathological sections of heart,lung,liver,and brain from the mice were carried out by H&E staining analysis.Furthermore,another set of expeimnent was also conducted in healthy mice,which were intravenously injected with irradiated and unirradiated aAPC cells,and flow cytometry was used to detect the number of aAPC cells in mice.ResultsSection one:Successful establishment of the hematological tumor modelsThe presence and distribution of tumor cells in mice was observed by small animal in vivo fluorescence imaging system.The tumors continued to develop and spread in the body during the experiment.The tumor cells in PBMC,bone marrow and spleen were also confirmed by flow cytometry.Finally,the mice eventually develop to morbidity and death due to the tumor progression.Section two:Evaluation of the anti-tumor effect of CAR-T cells in vivoThe tumor burden in mice after anti-CD-19 CAR-T cells treatment was significantly reduced compared with the control group.In addition,the survival of mice was also significantly prolonged by CAR-T treatments.Section three:Validation of the safety of CAR-T cell and aAPC cell in vivoThe body weight was no difference between CAR-T groups and control group.And no pathological sections showed significant organic lesions.Thus,there were no obviously adverse reaction after CAR-T treatment in this mice model.Moreover,there was no significant aAPC cell residue in the mice injected with aAPC cells after irradiation.Conclusion:1.Two Burkitt's lymphoma mouse tumor-bearing models that specifically expresses human CD 19 were successfully established.2.The anti-CD 19 CAR-T cells prepared by piggyBac the transposon transformation system and aAPC had a certain tumor-killing activity in mice and can prolong the survival time of mice.3.Anti-CD 19 CAR-T cells and irradiated aAPC cells had no obvious adverse reactions and safety issues.