Abstract 5242: In vitro cytotoxicity assays to support CAR T cell evaluation against solid tumors

Abstract CAR T cells are donor/patient-derived T cells genetically engineered to express a chimeric receptor against a specific tumor antigen (TA), which allows an MHC-independent recognition and targeting of tumor cells. While CART cell therapies have been tremendously successful against hematological cancers, with 6 T cell products being approved up to date from the FDA (Sengsayadeth S etal, EJHaem. 2022), the activity on solid tumors is more difficult. Solid tumors remain a unique challenge in part due to trafficking problems of CAR T cells, a hostile TME and heterogenous antigen expression, which limits CART cell capacity to find their target. Several strategies are being developed at the moment with the aim to overcome these hurdles, such as multispecific CART cells, co-therapy with checkpoint inhibitors, or inhibition of DNA methylation enzymes to prevent exhaustion. To offer an in vitro platform to assess the effectiveness of upcoming cellular therapies we developed several pipelines to allow the investigation of cytotoxic potential in 2D and 3D formats. We show here our results using a CART cell product expressing a coreceptor to enhance its activation potential upon recognition of the target. We next tested the capacity of these cells to kill solid tumor in vitro by coculturing with potential target cells cultured in a monolayer (2D). By measuring the luciferase expression of the target cells, we could track the cytotoxicity exerted by the effector cells. In vitro, the CART cells were able to kill in a dose dependent manner different solid tumor cell lines expressing the cognate antigen for the co-receptor. At Reaction Biology we have developed spheroid-based 3D killing assays in which target cells from solid tumor entities are pre-cultured to form spheroids prior to their coculture with effector cells. Using this technique, we challenged the capacity of these CART cells to recognize their antigen in a 3D format. This 3D assay system can be used as well in combination with co therapies such as PD1 inhibitors, in a sensitive and high throughput manner. Finally, we evaluated the therapeutic capacity of these CART cells in vivo in a subcutaneous THP-1 tumor model. Mice treated with these CART cells showed a marked reduction of tumor volume. In summary we show here a pipeline to characterize in vitro the effectivity of new T cell products, in particular those designed to overcome some of the hurdles against solid tumors. Citation Format: Carla N. Castro, Veronica Bergo, Holger Weber, Philipp Metzger. In vitro cytotoxicity assays to support CAR T cell evaluation against solid tumors abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 5242.