Stenger et al. evaluated the functionality of potential off-the-shelf CD19-CAR T cells from healthy HLA-mismatched donors lacking endogenous TCR. TCR knockout CD19-CAR T cells (TCR- CAR T) expressed co-stimulatory, co-inhibitory, and exhaustion markers, and displayed anti-CD19 activity that was comparable to TCR+ CAR T cells in vitro, but did not show alloreactivity. Both TCR+ and TCR- CAR T cells increased survival in PDX and NALM6 leukemia xenograft mouse models initially. However, TCR+ CAR T cells showed significantly improved long-term survival and in vivo persistence over TCR- CAR T cells, but with some alloreactivity.
Contributed by Shishir Pant
ABSTRACT: Anti-CD19 chimeric antigen receptor (CAR) T cells showed significant anti-leukemic activity in B-precursor acute lymphoblastic leukemia (ALL). Allogeneic, HLA-mismatched off-the-shelf 3rd-party donors may offer ideal fitness of the effector cells but carry the risk of graft-versus-host disease. Knockout of the endogenous T-cell receptor (TCR) in CD19-CAR-T cells may be a promising solution. Here, we induced a CRISPR/Cas9-mediated knockout of the TCRb-chain in combination with a 2nd-generation retroviral CAR transduction including a 4-1BB costimulatory domain in primary T cells. This tandem engineering led to a highly functional population of TCR-KO-CAR-T cells with strong activation (CD25, IFN-gamma), proliferation and specific killing upon CD19 target recognition. TCR-KO-CAR-T cells had a balanced phenotype of central memory and effector memory T cells. KO of the endogenous TCR in T cells strongly ablated alloreactivity in comparison to TCR-expressing T cells. In a patient-derived xenograft model of childhood ALL, TCR-KO-CAR-T cells clearly controlled CD19+ leukemia burden and improved survival in vivo. However, co-expression of endogenous TCR plus CAR led to superior persistence of T cells and significantly prolonged leukemia control in vivo, confirmed by a second in vivo model using NALM6 leukemia cells. These results point towards an essential role of the endogenous TCR for longevity of the response at the price of alloreactivity. In conclusion, anti-CD19 CAR-T cells with a CRISPR/Cas9-mediated TCR-KO are promising candidates for non-matched third-party adoptive T-cell transfer with high anti-leukemic functionality in the absence of alloreactivity, but long-term persistence in vivo is better in the presence of the endogenous TCR.