Identification of Healthy Donor Phenotypic and Functional Signatures That Predict Allogeneic, Hypoimmune CD19-Directed CAR T Cell Potency

Allogeneic, hypoimmune (HIP) CAR T cells may provide off-the-shelf CAR T cells that allow for expanded access for patients, overcome manufacturing failures, and provide an efficacious alternative to CAR T cells manufactured from heavily pre-treated T cells. However, donor T cells are the primary source of variability during production of allogeneic HIP CAR T cells, and may influence clinical efficacy. Previously, we reported a series of T cell functional assays that differentiate T cell potency as a means to select high-quality CAR T cell donors for clinical trials. Here, we explore donor phenotypic and functional signatures that are associated with desirable pre-clinical attributes for the generation of allogeneic HIP CD19-directed CAR T cells.

Human T cells from healthy donors were obtained by leukapheresis and used to generate allogeneic HIP CD19-directed CAR T cells by disrupting B2M, CIITA, and TRAC genes using Cas12b nuclease, and lentiviral transduction to overexpress CD47 and to express the CD19 CAR.Phenotype profiling (PBMC subsets, memory subsets, viability, exhaustion, and activation markers) was assessed on apheresis, CD4 or CD8 enriched subsets and post-CAR manufacturing. Generated CAR T cells were evaluated in vitro by repetitive tumor challenge (3x) performed over a 14-day period using the IncuCyte platform. Bulk cytokine analysis was measured by MesoScale Discovery (MSD). In vivo efficacy was evaluated using a systemic NALM-6 challenge in immune-deficient NSG mice at HIP CD19 CAR T cell doses of 5e5 and 5e6 cells per animal. Gene expression profiling was assessed on pre-production, post-CAR T cell manufacturing, and post-serial tumor challenge CD4+ and CD8+ T cells using the Nanostring nCounter CAR T panel. Functional and phenotypic attributes were used for weighted donor performance scoring. To do this, cell functional assays were defined to produce single, quantitative indices to enable the classification of CAR T cells based on functional performance. We assessed differences in CAR T cell performance and rank scored donor performance. Associations between donor attributes, phenotype/ functional T cell attributes and ranked donor performance were evaluated using Spearman's correlations.

Correlative comparison of phenotype and donor attributes in relation to weighted donor performance scores indicated that cellular subtypes at leukapheresis correlate with CAR T performance. Gene expression profiling shows that outperforming donors upregulate expression of mucosal associated invariant T (MAIT) cell canonical TCRb chain TRBV28. Outperforming donors have increased expression of Tc1/Th1 and Tc17/Th17 associated cytokines: IFNg, Il-17A, Il-17F, Il23, Il22, and have reduced expression of Tc2/Th2 associated cytokine Il5. Outperforming donors also showed reduced expression of MHC class II of alpha and beta heterodimers (HLA-DQA1 and HLA-DQB1). The information collected across phenotypic and functional comparisons will be compared with clinical performance.