During the 14-day in vitro expansion period prior to infusion, newly transduced CAIX-specific T cells shifted phenotype from CD4+ to CD8+ and maturation state from naïve to central effector (CE). The in vivo expansion potential of the cells correlated with the percentage of naïve cells at transduction and CE cells at day 14, suggesting that transduction of the naïve cells results in a more robust proliferative capacity of the CAR T cells.
Autologous T cells were genetically modified to express a chimeric antigen receptor (CAR) directed toward carboxy-anhydrase-IX (CAIX) and used to treat patients with CAIX-positive metastatic renal cell carcinoma. In this study, we questioned whether the T cell maturation stage in the pre-infusion product affected CAIX CAR expression and function in vitro as well as in vivo CAR T cell numbers and expansion. During the 14 days expansion of CAR T cells prior to administration, we observed shifts from a predominant CD4 to a CD8 T cell phenotype and from a significant fraction of naive to central effector T cells. Surface expression of the CAR was equally distributed among different T cell subsets and T cell maturation stages. During T cell culture days 14-18 (which covered patient treatment days 1-5), T cells demonstrated a decline in CAR expression level per cell irrespective of T cell maturation stage, although the proportion of CAR-positive T cells and CAR-mediated T cell effector functions remained similar for both CD4 and CD8 T cell populations. Notably, patients with a higher fraction of naive CD8 T cells at baseline (prior to genetic modification) or central effector CD8 T cells at 2 weeks of CAR T cell culture demonstrated a higher fold expansion and absolute numbers of circulating CAR T cells at 1 month after start of therapy. We conclude that the T cell maturation stage prior to and during CAR T cell expansion culture is related to in vivo CAR T cell expansion.