Allogeneic FLT3 CAR T Cells with an Off-Switch Exhibit Potent Activity against AML and Can Be Depleted to Expedite Bone Marrow Recovery

Abstract

Patients with relapsed or refractory acute myeloid leukemia (AML) have a dismal prognosis and limited treatment options. Chimeric antigen receptor (CAR) T cells have achieved unprecedented clinical responses in patients with B cell leukemias and lymphomas and could prove highly efficacious in AML. However, a significant number of patients with AML may not receive treatment with an autologous product due to manufacturing failures associated with low lymphocyte counts or rapid disease progression while the therapeutic is being produced. We report the preclinical evaluation of an off-the-shelf CAR T cell therapy targeting Fms-related tyrosine kinase 3 (FLT3) for the treatment of AML. Single-chain variable fragments (scFvs) targeting various epitopes in the extracellular region of FLT3 were inserted into CAR constructs and tested for their ability to redirect T cell specificity and effector function to FLT3 + AML cells. A lead CAR, exhibiting minimal tonic signaling and robust activity in vitro and in vivo, was selected and then modified to incorporate a rituximab-responsive off-switch in cis. We found that allogeneic FLT3 CAR T cells, generated from healthydonor T cells, eliminate primary AML blasts but are also active against mouse and human hematopoietic stem and progenitor cells, indicating risk of myelotoxicity. By employing a surrogate CAR with affinity to murine FLT3, we show that rituximabmediated depletion of FLT3 CAR T cells after AML eradication enables bone marrow recovery without compromising leukemia remission. These results support clinical investigation of allogeneic FLT3 CAR T cells in AML and other FLT3 + hematologic malignancies.

Immunotherapy in Acute Myeloid Leukemia: Where We Stand

“…To overcome some of these drawbacks related to the targeting of FLT3, one recent study reported the preclinical evaluation of an off-the-shelf CAR T cell therapy targeting FLT3 for the treatment of AML with reduced myelotoxicity while sparing anti-leukemic activity. Indeed, by using a surrogate CAR with affinity to murine FLT3, rituximab-mediated depletion of FLT3 CAR T cells after AML eradication enables bone marrow recovery without compromising leukemia remission ( 101 ).…”

Does lineage plasticity enable escape from CAR-T cell therapy? Lessons from MLL-r leukemia

“…Given the ongoing development of myeloid-directed CAR T cells for AML, this approach may be closest to clinical use, but combinatorial toxicity would have to be carefully considered. FLT3 CARs have been effective in preclinical studies [92] and are particularly relevant because of the high expression levels on pediatric MLL-r B-ALL [93−97] as well as myeloid leukemias [98−100]. The optimal strategy for simultaneously targeting FLT3 and CD19…”

Driving CAR T Stem Cell Targeting in Acute Myeloid Leukemia: The Roads to Success

“…Finally, AML intrinsic features and prior chemotherapy treatments may impact on the fitness of the underlying T-cell populations used for CAR engineering, challenging the successful manufacture of autologous CAR T-cells from AML patients [ 121 ]. Allogeneic CAR T-cells engineered to attenuate graft-versus-host disease and rejection may be employed to achieve faster and broader product availability [ 159 ]. A phase I clinical study is currently investigating the applicability of universal CD123 CAR T-cells in AML (NCT03190278).…”