Background Autologous CAR T cell therapies have revolutionized the treatment landscape in hematological malignancies. Using the patient’s own T cells for manufacturing, however, poses limitations on the widespread use of these therapies. Off-the-shelf allogeneic CAR T cells could potentially address these issues by using healthy donor T cells as starting material, consistency of product, immediate availability, and cost and convenience of scalable manufacturing. However, expansion and persistence of infused allogeneic CAR T cells may be limited by immune rejection. Immune “cloaking” strategies centered on deletion of β2-microglobulin can avoid rejection by CD8 T cells but may elicit strong NK cell rejection. Moreover, HLA Class II expression can be induced upon T cell activation to increase the risk of CD4 T cell rejection. Here, we propose an alternative approach to immune evasion by selectively targeting NLRC5 and RFX5, transcriptional regulators controlling expression of HLA molecules.
Methods CRISPR/Cas9 technology was used to knockout NLRC5, RFX5, B2M, CIITA, and/or TRAC. Survival of hypoimmunogenic cells was assessed in mixed lymphocyte reaction (MLR) assays with allogeneic T cells, NK cells, or PBMCs. For in vivo evaluation, mice were engrafted with human T cells and Raji tumor cells followed by administration of hypoimmunogenic CD19 CAR T cells, and CAR T cell persistence and tumor growth were monitored over time.
Results Deletion of NLRC5 and RFX5 resulted in substantial and stable downmodulation, but not complete ablation, of HLA Class I expression. RFX5 KO cells also exhibited downregulation of HLA Class II expression. NLRC5 KO and RFX5 KO T cells showed enhanced survival against allogeneic T cells but elicited only minor NK cell reactivity. When co-cultured with HLA-mismatched PBMCs, NLRC5 KO and RFX5 KO cells effectively mitigated rejection, whereas uncloaked control and B2M KO cells were eliminated by allogeneic T and NK cells, respectively. These findings were replicated in T cells expressing a CD19 CAR. Inactivation of NLRC5 or RFX5 did not impact CAR T cell phenotype or cytotoxic activity. In vivo, hypoimmunogenic CAR T cells demonstrated superior persistence and antitumor efficacy compared to uncloaked control CAR T cells in the presence of allogeneic T cells.
Conclusions Hypoimmunogenic CAR T cells can be successfully generated by targeted deletion of NLRC5 or RFX5, which reduces T cell rejection without triggering substantial NK cell rejection and does not affect CAR T cell function. The improved persistence of hypoimmunogenic allogeneic CAR T cells may increase the therapeutic efficacy of off-the-shelf products.
Statistics from Altmetric.com
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.