Human myeloid enhanced model systems: Tools for advanced evaluation of short-term CAR T cells and in vivo CAR T cell generation

Immunotherapies have emerged as a viable treatment option in cancer, by harnessing the natural ability of the immune system to eliminate tumor cells. Additional modification of the effector cells paved the way for novel strategies, such as equipping T cells with a chimeric antigen receptor (CAR) for efficient and target-specific tumor cell lysis. In particular, CD19CAR T cells showed tremendous results against B cell malignancies with currently four CD19CAR T cell products on the market. Despite the great successes CAR T cell manufacturing requires long ex vivo generation and cultivation time up to several weeks and therefore limits the application. Current efforts to reduce and avoid this limitation involves ex vivo manufacturing within few days and CAR T cell generation in vivo. This thesis evaluates these novel CAR T cell generation approaches in myeloid enhanced model systems to get a better understanding of potential safety risks and obstacles. Proof of concept for in vivo CAR T cell generation has been shown before with CD8 and CD4 receptor-targeted lentiviral vectors (CD8-LV and CD4-LV) in preclinical humanized mouse models. However, these studies were limited to humanized model systems that do not reflect the physiological myeloid cell composition in human accurately. That is why this thesis used the CD34+ stem cell humanized NSG-SGM3 (huSGM3) mouse model, which develops pronounced human innate immune cells including monocytes and macrophages, to evaluate in vivo CAR T cell generation. Intravenous (i.v.) injection of CD4-LV, CD8-LV or mixture of both vector types resulted in successful CD19CAR T cell generation in the blood of some mice. Importantly, CAR expression was restricted to the respective targeted CD4+ or CD8+ T cell subset without detectable off-target transduction. CD19+ target cell reduction in the system further underlined the presence of functional CD19CAR T cells in the periphery as well in various lymphocyte-residing and biodistribution-relevant organs. In addition, expansion of CAR T cells in the presence of tumor antigen confirmed presence and long persistence of CAR T cells in the spleen and transgene integration could be verified by qPCR analysis. Overall, in vivo CAR T cell generation was less efficient in huSGM3 compared to previous described CD34+ humanized NSG mice. Intriguingly, CD4-LV injected mice showed the least pronounced in vivo CAR T cell generation efficiency and correlated with a distinct plasma cytokine pattern associated with activated human myeloid cells. Reduced in vitro transduction of T cells with CD4-LV and CD8-LV in the presence of monocytes or macrophages identified these cells as potential barrier for in vivo CAR T cell generation. Strikingly, shielding CD4-LV and CD8-LV from phagocytosis, by using ß2M-/-, CD47high HEK-293T packaging cells for vector particle production, substantially improved CAR T cell generation in the co-culture. Furthermore, these modifications also improved in vivo CAR T cell generation in huSGM3 mice compared to conventional CD4-LV and CD8-LV. This was shown by higher vector copy numbers, more pronounced CD19+ cell reduction in the spleen and bone marrow for both shielded vector types and in addition by higher CAR T cell numbers in the blood for shielded CD4-LV. Reducing ex vivo CAR T cell generation time is another attractive approach to refine manufacturing. In this thesis CD19CAR T cells generated within 3 days (short-term CAR T cells) using lentiviral vector incubation were evaluated for cellular composition and potential to induce cytokine release syndrome (CRS), a common side effect in CAR T cell therapy. Characterization of short-term CAR T cells after production revealed that most of the cells were vector particle-bound and only a minority was CAR positive. A designed co-culture model with tumor cells and monocytes demonstrated tumor cell specific cytotoxicity for short-term CAR T cells, which showed substantial CAR expression after 24h of co-culture. Moreover, the co-culture model revealed high release of CRS-relevant cytokines including IL-6, IFN-γ, TNF-α, GM-CSF, IL-2 and IL-10 during the killing assay. Using NSG-SGM3 mice with high tumor burden, i.v. administration of high numbers of short-term CAR T cells showed severe acute events within 24h. This included body signs of ill-being, temperature and weight drop and systemic cytokine elevation of human IFN-γ, TNF-α, IL-2 and IL-10 by short-term CAR T cells and murine MCP-1, IL-6 and G-CSF by the mouse system. Taken together, this thesis highlights the potential of sophisticated model systems to evaluate possible roadblocks and safety risks of novel CAR T cell therapy manufacturing approaches. They allowed the identification of human monocytes and macrophages as potential barrier for in vivo CAR T cell generation and highly supports the implementation of phagocytosis shielding in targeted LVs to reduce innate immune response for this approach. Moreover, such model systems revealed the potential of short-term CAR T cells to induce severe acute CRS and emphasize a careful approach in the clinic and further assessment on these model platforms