Assessing donor heterogeneity in CAR-T cells with massively parallel mixed lymphocyte reactions

Donor heterogeneity is a major source of variation in CAR-T cell functions which leads to inconsistencies in both clinical efficacy and pre-clinical studies to enhance CAR-T cell functions. However, assessing T cell donor heterogeneity with a large sample size is costly and time-consuming. Here, we developed a Massively Parallel - Mixed Lymphocytes Reaction (MP-MLR) assay that allows pooling and simultaneously testing of CAR-T cells from multiple human donors. Combining with pooled genetic perturbation technologies, we conducted pooled screening in more than 50 T cell donors for enhanced CAR-T cell proliferation under repetitive tumor stimulations, with a library of candidate genetic perturbations proposed to enhance CAR-T or T cell functions. While we identified genetic perturbations with consistent effect on CAR-T cells across donors, the majority of perturbations showed various levels of donor dependencies, and this observation was confirmed with arrayed validation of selected perturbations on specific donors. Furthermore, we performed multi-parameters pooled screening in-vitro and in-vivo for systematic discovery of generalizable and donor specific genetic perturbations enhancing CAR-T cell pre-clinical therapeutic efficacies. Overall, we presented an efficient and cost-effective strategy for assessing donor variabilities in CAR-T cells, predicting patient specific effects of genetic perturbations on CAR-T cells efficacies, and potentially identifying of personalized optimal genetic perturbation for enhancing CAR-T cell functions.