Assistant Professor University of Illinois at Urbana Champaign Urbana, Illinois, United States
Introduction: CAR T therapy is an emerging cancer treatment in which the patients’ own T cells are engineered ex vivo towards expressing chimeric antigen receptors and thus specifically recognizing and attacking the cancer cells. Though CAR T therapy has proven to be effective in treating certain types of blood cancers, challenges persist in its efficacy when targeting solid tumors. One of the reasons is that CAR T cells become exhausted in the tumor microenvironment which contains multiple cytokines, chemokines and metabolites that inhibit CAR T. To improve the in vivo efficacy of CAR T therapy, here we propose a combination between CAR T therapy and dendritic cell vaccine. By conjugating target of CAR T cells to the surfaces of dendritic cells, the activation and proliferation of CAR T cells will be enhanced.
Methods: • Generation of mouse anti-mouse CD19 CAR T cell In brief, primary CD8 T cells were isolated from spleens of BALB/C mice. The T cells then undergo in vitro activation and expansion before being transduced by lentiviral vector. Transduced cells were then screened by puromycin and enriched before use. • Generation of CD19 conjugated dendritic cell Dendritic cells were differentiated from bone marrow cells of BALB/C mice. At day 6 after differentiation, Ac4ManNAz was added into culture medium to metabolically label the cells. Cells were then washed before incubating with DBCO-CD19. • In vitro coculture of DC and CAR T 1*10^5 DC with different pretreatment were seeded into 96 well plate, then corresponding number of CAR T cells without Dynabeads were added. Culture medium was free of any cytokines. Coculture lasts overnight. • In vitro killing assay Calcein AM was used to stain live A20 cells. After coculture, cells were centrifuged, the supernatant from each well was collected and fluorescence intensity was measured with microplate reader.
Results, Conclusions, and Discussions: • Figure 1 demonstrates the successful transduction of murine CD8 T cell by detecting the expression of F(ab')₂ fragment on the CAR construct. • In figure 2 and 3, the functionality of CAR T cells was verified. In activation assay, the CAR T cells were cultured with soluble CD19 protein. Compared with control T cells, the CAR T shows an enhanced activation when exposed to their target. Here two markers CD69 and CD40L were used for evaluating the T cell activation level. In cytotoxicity assay, the CAR T cells were cocultured with mouse lymphoma A20 tumor cells, cytotoxicity was calculated by measuring the Calcein AM signal. • In figure 4, the CD19 protein on the surface of dendritic cells was detected. Result from flow cytometry indicates that metabolic labeling enables CD19 conjugation through bioorthogonal chemistry reaction between azide and DBCO groups. • In figure 5, the activation effect of dendritic cells conjugated with CD19 on CAR T cells was assessed through flow cytometry. More specifically, CD69 expression level was measured among the CD3+CD8+ live cell population. Result indicates an improved activation status of CAR T cells cocultured with dendritic cells conjugated with CD19. • In figure 6, Calcein AM labeled A20 tumor cells were added into the coculture system. The functionality enhancement of CAR T cells were indicated by increase in cytotoxicity. Different coculture ratio was applied, the efficacy of CAR T could be best enhanced when CAR T: A20: DC ratio = 2:1:2. o Conclusions Through in vitro experiments we demonstrated an enhancement in CAR T activation status and cytotoxicity induced by engineered dendritic cell vaccine. This boosting effect is achieved by chemically conjugating the target protein onto the surfaces of dendritic cells so that CAR T cells not only recognize the target but could also be further activated by DC.
The in vivo combination of DC vaccine as a prophylactic approach and CAR T cell as a therapeutic approach is a promising direction, which we hope to explore next.
