Abstract 5184: Real-time cytotoxicity assays as a pre-clinical screening tool for LGR5-targeting CAR-T cells for treatment of solid tumors

Current challenges for CAR-T therapies in solid tumors relate to target specificity and T cell penetration of the tumor. A key determinant in CAR-T selection for clinical trials is the potency and persistence of the selected CAR-T to kill cancerous cells. This is often difficult to determine across different donors and batches. Additionally, these characteristics can be difficult to determine in vitro in a rapid, high-throughput manner. In this study we have utilized real-time impedance cytotoxicity assays to screen LGR5-targeting CAR-T cells. LGR5 is a well described cancer stem cell antigen implicated in tumor initiation and metastasis, and prognosis of colon, ovarian and neuroblastoma cancers. Impedance measurements are taken continuously, providing comprehensive real-time information about CAR-T cell efficiency over time, which is not achievable with single timepoint cytotoxicity assays. Through this validation process we have been able to concurrently test different Effector to Target (E:T) ratios and different CAR constructs to accurately and directly compare CAR-T killing efficiency. This readout is a key indicator of CAR-T potency. Similar to inhibitory concentration 50 (IC50) or lethal dose 50 (LD50) data, CAR-T potency can be expressed in a measure as kill time 50 (KT50), and represents the time required for the CAR-T to stimulate cytolysis in 50% of the target cancer cells. KT50 data was key to the selection of our lead clinical candidate; CNA3103. In addition to selection of our lead candidate, real-time impedance technology has allowed us to screen a broad range of LGR5-expressing cancer types. Our data indicates that CNA3103 can effectively target colon, neuroblastoma, and ovarian cancer cells in vitro. Real-time impedance assay has allowed us to analyze cancer cells with different levels of LGR5 expression to confirm the specificity of our CAR-T cells and to develop an understanding of how LGR5 expression levels might impact cancer cell killing. The data also indicates that our LGR5 targeting CAR-T cells have the potential to repeatedly kill, i.e. the same CAR-T cell is able to move on from one cancer cell to another and remain cytotoxic. Taken together, real-time cytotoxicity assays provide valuable pre-clinical data, enabling the selection of CNA3103 as our LRG5-CAR-T clinical candidate. This technology may also assist in patient and dose selection within a clinical trial. Citation Format: Emma J. Thompson, Veronika Bandara, Timothy Sadlon