In Vivo Production Of CAR-T Cells Using Fusogenic Nanovesicles

Chimeric antigen receptor（CAR）T cell therapy is a revolutionary immune cell therapy,which has achieved great success in the treatment of blood hematological malignancies.Although CAR-T cell therapy has developed rapidly in clinics,the problems such as high-priced treatment,long preparation periods,and obvious side effects limit the application of CAR-T cells As a personalized treatment scheme,CAR-T cell therapy needs to repeat several steps for each patient,such as T cell isolation,CAR gene transduction,T cell activation,expansion,and reinfusion to the patient.Therefore,the manufacture of CAR-T cells takes a long time for preparation,and has high requirements for medical staff and facilities.In addition,side effects,including cytokine release syndrome,in CAR-T cell therapy affect patient safety greatly.To deal with these problems,it is necessary to develop a method that can quickly prepare autologous CAR-T cells and reduce the requirements for personnel and facilities.Moreover,it is important to reduce the side effects of CAR-T cell therapy and ease the burden on patients.Extracellular vesicles are cell-derived membrane-based particles with low immunogenicity and good biocompatibility.As carriers for intercellular signaling,extracellular vesicles can carry proteins,nucleic acids,and small molecules,and have various advantages,such as stability in circulation.For targeted drug delivery,modified extracellular vesicles can increase tumor targeting and achieve better delivery of chemotherapy drugs.Furthermore,membrane proteins can be delivered from extracellular vesicles to the cell surface via membrane fusion.In this research,we report a nanovesicle-based T cell membrane fusion system to deliverαCD19 CAR to prepareαCD19 CAR-T cells in vivo.It is mainly divided into the following two parts:1.T cells are important immune cells in the body and play an important role in both cellular and humoral immunity.Engineering T cells is generally carried out using viruses or electroporation.To develop a method for efficient transduction of T cells,we construct T cell membrane fusogen by engineering the FAST protein p14 and measles virus-related proteins（MVFP,H protein,and F protein）.Our results show that T cell membrane fusogen could induce the fusion of T cell membrane fusogen-expressing cells and T cells in vitro.Cell-derived nanovesicles（NVs）carrying T cell membrane fusion proteins（FuNV）were able to induce the fusion of NVs and T cells both in vitro and in vivo.In addition,the injection of FuNV did not induce significant hepatotoxicity or other systemic toxicity.2.Given the time-consuming and highly demanding manufacture of CAR-T cells in vitro,we prepared nanovesicles carrying T cell membrane fusion protein andαCD19 CAR simultaneously(FuNV_CAR)based on FuNV.FuNV_CAR can deliverαCD19 CAR to T cells to prepare CAR-T cells both in vitro and in vivo.The results of this study demonstrate that FuNV_CAR can generateαCD19 CAR-T cells in vitro,which induce cytotoxicity to B lymphoma cells specifically.αCD19 CAR-T cells can be prepared by injection of FuNV_CAR in vivo,and significantly inhibit B lymphoma growth without cytokine release syndrome.In combination with immune checkpoint antibody therapy,FuNV_CAR could inhibit tumor growth more effectively.The preparation of CAR-T cells by CAR protein delivery in vivo could effectively inhibit tumor growth and avoid the side effects of traditional CAR-T cell therapy,which provides a novel approach for CAR-T cell application.