Murine Allogeneic CAR-T Cells Integrated Before or Early After Posttransplant Cyclophosphamide Exert Anti-Tumor Effects.

Relapse limits the therapeutic efficacy both of chimeric-antigen-receptor (CAR)-T-cells and allogeneic hematopoietic cell transplantation (allo-HCT). Patients may undergo these therapies sequentially to prevent or treat relapsed malignancy. However, direct integration of the two therapies has been avoided over concerns for potential induction of graft-versus-host disease (GVHD) by allogeneic CAR-T-cells. We have shown in murine T-cell-replete MHC-haploidentical allo-HCT that suppressive mechanisms induced immediately after post-transplantation cyclophosphamide (PTCy), given on days +3/+4, prevent GVHD induction by alloreactive T-cells infused as early as day +5. Therefore, we hypothesized that allogeneic CAR-T-cells given in a similarly integrated manner in our murine MHC-haploidentical allo-HCT model may safely exert anti-tumor effects. Indeed, allogeneic anti-CD19 CAR-T cells given early after (day +5) PTCy or even prior to (day 0) PTCy cleared leukemia without exacerbating the cytokine release syndrome occurring from the MHC-haploidentical allo-HCT or interfering with PTCy-mediated GVHD prevention. Meanwhile, CAR-T-cell treatment on day +9 or +14 was safe but less effective, suggesting a limited therapeutic window. CAR-T-cells infused before PTCy were not eliminated, but surviving CAR-T-cells continued to proliferate highly and expand despite PTCy. In comparison with infusion on day +5, CAR-T-cell infusion on day 0 demonstrated superior clinical efficacy associated with earlier CAR-T-cell expansion, higher phenotypic CAR-T-cell activation, less CD4+CD25+Foxp3+ CAR-T-cell recovery, and transcriptional changes suggesting increased activation of CD4+ CAR-T-cells and more cytotoxic CD8+ CAR-T-cells. This study provides mechanistic insight into PTCy's impact on graft-versus-tumor immunity and describes novel approaches to integrate CAR-T-cells and allo-HCT that may compensate for deficiencies of each individual approach.