Hypoimmune FK binding protein 12-knockout CD19 CAR T cells achieve deep tissue B-cell depletion and control Nalm6 tumors in immunosuppressed humanized mice

Abstract

Autologous chimeric antigen receptor (CAR) T-cell therapy in solid-organ transplant recipients on life-long systemic immunosuppression has so far been disappointing. The mTOR inhibitors tacrolimus (Tac) and rapamycin (Rapa) are among the most common immunosuppressive drugs. While they prevent organ allograft rejection, they also suppress the efficacy and persistence of the CAR T-cell product. A reduction in immunosuppression is usually recommended before leukapheresis and after infusion of the final CAR therapeutic to facilitate CAR T-cell activity. However, this reduction jeopardizes allograft survival and multiple graft failures have been reported. Here, we report the engineering of allogeneic, human CD19 CAR T cells that are resistant to mTOR inhibitors through the knockout of FK-binding protein 12 (FKBP-KO). Both HLA-replete WT-FKBP-KO and hypoimmune (HLA class I- and II-depleted and CD47-overexpressing, HIP) CD19 CAR T cells maintained effective in vitro killing capacity of benign human B cells and malignant Nalm6 tumor cells when incubated with high Tac and Rapa concentrations. CAR T cells without the FKBP-KO failed to kill both targets in the presence of Tac or Rapa. HIP-FKBP-KO CAR T cells were able to fully evade cytotoxicity from primed allogeneic T cells, natural killer (NK cells), and macrophages. When injected into immunosuppressed Nalm6-bearing humanized mice, only the HIP-FKBP-KO CAR T cells achieved persistence and deep tissue depletion of CD19⁺ cells over a 25-day study period, whereas the WT-FKBP-KO CAR T cells diminished in number and efficacy. Allogeneic HIP-FKBP-KO CAR T cells may treat post-transplant lymphoproliferative disease effectively in solid organ transplant recipients maintained on immunosuppression.