CD8 Receptor-Targeted Lentiviral Vectors – an Approach for the in vivo Generation of Chimeric Antigen Receptor (CAR) T Cells

Gene therapeutic applications have gained substantial significance in modern medicine, especially for the treatment of cancer diseases. Genetically engineered T cells that express a chimeric antigen receptor (CAR) have been shown to mediate impressive anti tumoral efficacy in patients suffering from B cell malignancies. In 2017, the first CAR T cell product was approved in the United States (U.S.). However, cell selective gene delivery still represents a big hurdle, making ex vivo gene delivery indispensable that is accompanied by complex efforts and high costs due to the personalized treatment. Receptor-targeted lentiviral vectors mediate selective gene delivery into a certain cell type and represent a powerful tool for the in vivo gene transfer. This thesis investigates the in vivo generation of CAR T cells in small animal models using a CD8-targeted lentiviral vector (CD8-LV). CD8-LV has been generated before by pseudotyping lentiviral vectors with modified Nipah virus glycoproteins displaying an anti-human CD8-targeting domain. In this thesis, selective in vivo reporter gene delivery into CD8+ lymphocytes was demonstrated upon systemic administration of CD8-LV into mice engrafted with human peripheral blood mononuclear cells (PBMC). Thereby, reporter gene expression exclusively within the CD8+ cells proved the highly selective targeting of CD8-LV. In vitro generation of CAR T cells upon transduction of PBMC with CD8-LV transferring a CD19 specific chimeric antigen receptor was shown, and functionality of these generated CAR T cells was demonstrated. They selectively expanded upon antigen stimulus and specifically killed CD19+ target tumor cells in vitro. CD8-LV(CAR) administration into mice resulted in the in vivo generation of CAR T cells with remarkably high frequencies of CAR positive cells. Higher frequencies of transgene-positive and CD8-positive cells compared to reporter gene transfer indicated selective CAR T cell expansion in vivo. Importantly, functionality of in vivo generated CAR T cells was demonstrated when CD19+ target cells had been eliminated. Moreover, CD19+ cells were identified as antigen stimulus triggering antigen-specific CAR T cell proliferation. Phenotype analysis of CAR T cells by surface marker analysis revealed the presence of diversely differentiated CAR T cells, which is highly preferable in terms of generating a pool of CAR T cells with various effector functions and proliferative capabilities. Furthermore, anti-tumoral efficacy was evaluated in xenograft mice engrafted with human tumor cells. Although tumor outgrowth was not prevented, these CAR T cells demonstrated killing activities against CD19+ B cells and emigrated to various organs. Showing organ-specific subset distribution of diversely differentiated CAR T cells, highest levels of effector CAR T cells were observed at the tumor site. This thesis highlights the potential of CD8-LV to genetically engineer CD8 T cells in vivo. Selective gene transfer and functionality of in vivo generated human CAR T cells represent encouraging data to build on for further investigations in translational research. Pursuing receptor-targeted LVs for clinical application as an alternative approach of CAR T cell generation opens up an attractive possibility to tremendously simplifying CAR T cell therapy. In conclusion, CD8-LV represents a promising tool for the in vivo CAR T cell generation with the potential to transform personalized CAR T cell therapy into a broad applicable treatment option