Lipid Nanoparticles Produce Chimeric Antigen Receptor T Cells And Chimeric Antigen Receptor Macrophages In Vivo

Chimeric antigen receptor T-cell(CAR-T)therapy has achieved remarkable success in the treatment of hematological malignancies,with several products currently approved by the FDA for acute B-cell leukemia(B-ALL),large B-cell lymphoma and multiple myeloma.However,there are two limitations for the clinical application of CAR-T technology: a complicated preparation process ex vivo and cytokine release syndrome(CRS).Currently,obtaining CAR-T cells requires a complex process and strict quality control.Additionally,the preparation of CAR-T cells for clinical applications requires customization and following GMP for each patient.Another challenge for CAR-T therapy is cytokine release syndrome(CRS)caused by IL-6.In this study,we constructed a lipid nanoparticle system modified by CD3 antibody on the surface,loading with the plasmid containing the combination gene of interleukin 6 short hairpin RNA(IL-6 sh RNA)and CD19-CAR(Anti CD3-LNP/CAR19+sh IL6).The system targeted T cells by the mediation of CD3 antibody and stably transfected T cells under the influence of the nuclear localization peptide MTAS-NLS to transform them into CAR-T cells with IL-6 knockdown,thus killing CD19-highly expressed leukemia tumor cells and reducing CRS caused by IL-6.In vivo experiments showed that Anti CD3-LNP/CAR19+sh IL6 could stably transfect T cells and produce CAR-T within 90 days to kill the tumor.This significantly prolonged the survival time of leukemia model mice and demonstrated the prepared LNP exhibited the same antitumor effect as the traditional CAR-T cells prepared ex vivo.At the same time,IL-6 expression was silenced,which would be helpful to reduce the CRS and improve the safety of CAR-T therapy.This technology could avoid the complex preparation process ex vivo and strict quality control of the traditional CAR-T technology,reduce the technical barrier and production costs.This method improves the convenience of using CAR-T technology and is helpful in further promoting the clinical application of CART.Chimeric antigen receptor T cell(CAR-T)therapy has shown a good therapeutic effect on hematological tumors,however,its therapeutic effect on solid tumors is limited.At this time,based on CAR-T and CAR-NK therapy,the chimeric antigen receptor-macrophage(CAR-M)began to develop rapidly,and showed a good therapeutic effect on solid tumors.However,the current production process of CAR-M ex vivo is complex and costly,and the CAR-M cells produced cannot be proliferated in vivo.The two factors have seriously limited its clinical application.Thus,we got inspiration from the research on the direct production of CAR-T cells in vivo,and used the lipid nanoparticles(LNPs)delivery system again,loaded with plasmid containing Trop2-CAR(LNPs/CAR Trop2),which was used to directly produce CAR-M cells in vivo.In addition,macrophages can present antigens of phagocytosed tumor cells to T cells and activated the CD8+T cells,which could stimulate CD8+T cells to kill tumor cells.The results showed that following i.v.injection,the system stably transfected macrophages in vivo to transform them into CAR-M cells,which effectively penetrated solid tumors and killed Trop2-highly expressed tumors.With this method,LNPs/CAR Trop2 CAR-M cells could be directly produced in vivo after LNPs injection,avoiding the current complex production process ex vivo.The LNP Nano-delivery system utilized the "disadvantage" of being swallowed by mononuclear macrophages in blood,liver,spleen,lung and tumor tissues,and turn it into the "advantage" of being able to produce CAR-M cells,which solves the problem of preparation of CAR-M ex vivo,improves the convenience of using CAR-M technology,and promotes its clinical application.In conclusion,in this study,aiming at the challenges in the clinical application of CART and CAR-M therapy,such as complicated preparation process in vitro,potential cytokine release syndrome and limited therapeutic effect of solid tumor,we designed lipid nanoparticles to produce IL-6 knocked down chimeric anti-receptor T cells or chimeric antigen receptor macrophages in vivo.The Anti CD3-LNP/CAR19+sh IL6 could target T cells through the mediation of CD3 antibody,and produced CAR-T cells with IL-6 knockdown in vivo,thus killing tumor cells with high CD19 expression and reducing CRS caused by IL-6.In addition,the LNP drug delivery system(LNPS/CARTrop2),could directly produce CAR-M cells in vivo and kill tumor cells with high expression of Trop2 antigen.This technology could avoid the complicated ex vivo preparation process and strict quality control of traditional CAR-T/CAR-M technology,reduce technical barriers and production costs,and is helpful to promote the clinical application of CAR-T/CAR-M.