Background
The duration of T cell persistence post infusion is crucial for the success of adoptive cell therapy (ACT). Despite exhibiting full functionality, insufficient autologous or engineered T cell persistence can hinder an effective anti-tumor response. Consequently, T cells’ persistence and migratory characteristics play pivotal roles in determining the efficacy of immune-oncology products against tumors. There is a lack of widely accepted methods for quantitatively assessing the persistence of cell therapy products in ex-vivo systems for preclinical evaluation. Patient-derived xenografts and organoids, requiring weeks to months for establishment, are impractical in clinical settings and lack the scalability necessary for research and development. In contrast, tumoroids derived from patient tumors with minimal manipulation, preserve spatial heterogeneity, enhancing their clinical relevance. Adopting Nilogen’s ex-vivo tumoroid platform, we have developed a persistence assay for cell therapy products targeting solid tumors.
Methods
The assay involves exposing chimeric antigen receptor T cells (CAR-Ts) to fresh tumoroids expressing the same target over multiple cycles, assessing cytotoxic efficiency, and monitoring functional changes in the engineered CAR-Ts with repeated encounters. The interaction between CAR-Ts and tumoroids is captured through time-lapse imaging and analyzed using high-content imaging and flow cytometry-based techniques. An AI-driven algorithm compares initial and subsequent challenge datasets. For evaluating persistence of T cell function, quantification of clustering speed, aggregation timing, and tumor cell killing efficiency was considered. Flow cytometry evaluates the relative expression of T cell activation and exhaustion markers, correlating them with CAR-T persistence.
Results
A progressive decline in T cell persistence and functionality, accompanied by an increase in exhaustion marker expression, was evident with each successive challenge. In ACT, the limited replicative capacity of T cell receptors (TCRs) impeded these cells’ long-term persistence and expansion in patients, affecting their enduring therapeutic efficacy. Notably, TCR sequencing revealed the emergence of monoclonal enrichment of rare clonotypes, potentially those with superior proliferative potential using Nilogen’s tumoroid platform.
Conclusions
These findings highlight the utility of the tumoroid-based cell therapy persistence assay in optimizing and advancing modified cell therapy products during preclinical development.
Ethics Approval
Ethics approval was obtained through Chesapeak IRB (Pro00014313) which determined ‘Using the Department of Health and Human Services regulations at 45 CFR 46, the IRB determined that this research project does not constitute human subject research and, therefore, does not require IRB oversight.’ Full informed consent was obtained for each tissue used in this study.