ID #601 Developing in vitro assays to track CAR T cell treatment efficacy precisely and accurately

Abstract

Chimeric Antigen Receptor (CAR) T-cell therapy harbours great potential for treatment of currently incurable solid cancers, including paediatric brain cancers. Translation of this immunotherapy to solid tumours is complicated by a lack of assays to precisely measure tumour infiltration of CAR T-cells and hence, intratumoural efficacy. Additionally, monitoring of the tumour through repeated biopsies is highly invasive and access to solid tumour sites is often restricted. We are addressing these limitations through the development of a liquid biopsy-based assay to simultaneously profile cell-free DNA (cfDNA) from both tumour and CAR T-cells. This will provide longitudinal insights on the expansion kinetics of CAR T-cells as well as tumour burden and evolution. Furthermore, in-depth analysis of fragment size will allow for distinction between longer circulating cell-free CAR (cfCAR) DNA and shorter tumour-infiltrating cfCAR DNA, allowing for assessment of intratumoural activity. For the purpose of in vitro testing of this assay, we have incorporated complex 3D culture models to better mimic the barriers to T-cell infiltration that exist in vivo. This includes the use of assembloids consisting of tissue-relevant stem cell-derived organoids combined with tumour spheroids. These are then co-cultured with CAR T-cells. To confirm the utility of these models for detecting circulating tumour DNA (ctDNA), media was harvested from patient-derived tumouroids established from paediatric glioblastoma tumour samples. ctDNA was isolated from the culture medium, and mutations consistent with clinical reports from the corresponding primary tumours were confirmed by digital droplet PCR (ddPCR). Using this approach, we are now using cfDNA extracted from conditioned media as a surrogate to develop and test our NGS-based assay for detecting ctDNA and cfCAR DNA. Analysis of cfDNA from our 3D models may provide an alternative resource for tumour diagnosis, preclinical therapeutic screening, and personalised treatment selection, while reducing the need for repeated invasive tissue biopsies.