| Background and objectiveDiffuse large B cell lymphoma(DLBCL)is the most common type of non-hodgkin lymphoma(NHL),accounting for approximately 30-60%of its incidence.To date,the combination of rituximab and multi-drug chemotherapy has been recognized as the standard treatment for NHL.Patients can achieve long-term survival and high reactivity,but up to 40%of patients will develop recurrent or refractory disease.In recent years,chimeric antigen receptor T-cell(CAR-T)therapy has attracted more and more attention,and has achieved remarkable results in the treatment of hematological malignancy.IL-21 is a pleiotropic cytokine with functions of immune regulation and immunotherapy,which can act on a variety of immune cells.It can enhance the anti-tumor effect of T cells,NK cells and NKT cells,and induce the apoptosis of B cells and inhibiting the formation of tumor blood vessels in endothelial cells.Previous studies have shown that local expression of IL-21 in tumor sites can enhance innate and acquired T cell immunity.PD-1 is expressed on the surface of T cells and primary B cells,while tumor cells have high expression of PD-L1,which leads to programmed death of T cells through the combination of PD-1/PD-L1,so that tumor cells can escape the surveillance of the immune system and achieve immune escape.Studies have shown that most tumors respond to blocking PD-1/PD-L1 binding.This project intends to co-express IL-21 and PD1-Fc fusion proteins on CD 19 CAR-T(CAR19-T)cells.the extracellular area of human PD-1 is connected with the Fc segment of IgG1 to form fusion protein,which can block the function of PD-1/PD-L1,and bind with the Fc receptor on the surface of macrophages and NK cells to stimulate the ADCC,and perform multiple anti-tumor effects to enhance the efficacy of CAR19-T cells in killing tumor cells,it is intended to provide a new CAR-T cell therapy for recurrent or refractory DLBCL.MethodsThis project is to construct three kinds of recombinant plasmids that can express the target gene:PEF-KCAR19-IL21,PEF-KC AR19-PD1 Fc and PEF-KCAR19-IL21-PD1Fc based on the second-generation CAR targeted at CD19 and using genetic engineering technology.Four kinds of CAR-T cells were manufactured using the third-generation packaging system of lentivirus.The killing effect of CAR-T cells on DLBCL cell lines was detected by cell killing experiment(LDH method),and the Subcutaneous xenograft tumor animal model of diffuse large B cell lymphoma was constructed to detect the effect of CAR-T cells in vivo.Results1.Four types of CAR-T cells were successfully manufactured:CAR19-T,CAR19-IL21-T,CAR19-PD1Fc-T and CAR19-IL21-PD1Fc-T cells,with the CAR19 positive rates between 40%and 60%post corresponding lentivirus infection.2.DLBCL cell lines OCI-Ly8 and OCI-Ly3 were used as the target cells,four different types of CAR-T cells were co-cultured with the target cells respectively,and the results show that all types of CAR-T cells can effectively kill the target cells.The maximum killing efficiency of all types of CAR-T cells to OCI-LY8 and OCI-LY3 was close to 90%and 80%respectively3.In the subcutaneous xenograft tumor mouse models of using OCI-Ly8 and OCI-Ly3 target cells,among all five groups including the non-treatment group,the CAR19-IL21-T and CAR19-IL21-PD1Fc-T cells treatment groups resluted in better therapeutic benefit against the established tumors.While CAR19-T and CAR19-PD1Fc-T cells can only suppressed the tumor growth.ConclusionIn summary,four types of CAR-T cells were generated in this study including CAR19-T,CAR19-IL21-T,CAR19-PDlFc-T and CAR19-IL21-PD1Fc-T cells,and preliminary studies of these cells functions in vivo and in vitro were conducted.The results showed that CAR19-IL21-T and CAR19-IL21-PD1Fc-T cells had stronger anti-tumor effects,which may potentialy provide a new scheme for CAR-T therapy and laying the foundation for further investigation. |
PDF Full Text Request