Nonclinical Safety Assessment of AMG 553, an Investigational Chimeric Antigen Receptor T-Cell Therapy for the Treatment of Acute Myeloid Leukemia

Abstract

FMS-like tyrosine kinase 3 (FLT3), a tyrosine-protein kinase involved in hematopoiesis, is detectable on the cell surface of approximately 80% of leukemia isolates from adult patients with acute myeloid leukemia (AML). AMG 553 is an investigational chimeric antigen receptor (CAR) T-cell immunotherapy for the treatment of AML. FLT3 expression analysis and in vitro and in vivo studies were leveraged to evaluate the nonclinical safety of AMG 553. Cynomolgus monkeys administered autologous anti-FLT3 CAR T cells demonstrated no evidence of CAR T-cell–mediated toxicity, expansion, or persistence, likely due to restricted cell surface FLT3 protein expression in healthy animals. This highlights the limited value of such in vivo studies for safety assessment of the CAR T-cell modality when directed against a target with restricted expression. To complement these studies and directly evaluate the potential toxicities of eliciting T cell–mediated cytotoxicity against cells with surface expression of FLT3 protein in vivo, data from cynomolgus monkey toxicology studies with two bispecific T cell engager (BiTE®) molecules targeting FLT3 were leveraged; findings were consistent with the targeted killing of bone marrow cells expressing cell surface FLT3. Potential AMG 553-induced cytotoxicity was assessed against a wide range of normal human primary cells and cell lines; cytotoxicity was observed against FLT3-positive AML cell lines and a percentage of primary bone marrow CD34+ cells. In conclusion, the nonclinical safety data suggest that AMG 553 can target FLT3 protein on AML cells while only affecting a percentage of normal hematopoietic stem and progenitor cells, supporting clinical development.

A novel approach for relapsed/refractory FLT3mut+ acute myeloid leukaemia: synergistic effect of the combination of bispecific FLT3scFv/NKG2D-CAR T cells and gilteritinib

“…Previously, a phase 1 clinical trial revealed no grade 3 or above adverse events related to NKG2D CAR T cell therapy in patients with relapsed/refractory AML [43], indicating that it is safe targeting NKG2DLs in AML patients. Clinical data regarding the safety of FLT3-CAR T cells are currently not available, but a nonclinical safety assessment demonstrated that FLT3-CAR T cells only affect a small percentage of normal haematopoietic stem and progenitor cells [44], which is in agreement with our findings. In contrast, other studies have reported that FLT3-CAR T cells do not deplete HSCs and that HSC differentiation is preserved [17].…”

Potent preclinical activity of FLT3-directed chimeric antigen receptor T-cell immunotherapy against <i>FLT3</i>- mutant acute myeloid leukemia and <i>KMT2A</i>-rearranged acute lymphoblastic leukemia

“…We demonstrate potent in vitro and in vivo anti-leukemia efficacy against FLT3-mutant AML and KMT2A-R ALL cell lines, as well as robust FLT3CARTmediated eradication of leukemia in vivo in several PDX models of pediatric or young adult FLT3-mutant AML, FLT3 wild-type AML, and KMT2A-R ALL. Consistent with other studies of FLT3-directed CAR T cell immunotherapies, [38][39][40][41][42] our FLT3CART showed excellent activity against FLT3-ITD AML via in vitro cytokine production and viability metrics, as well as in vivo curative effects and long-term animal survival. We were intrigued to discover that flow cytometric FLT3 surface antigen site density was not overtly different in our tested FLT3-mutant vs non-mutant AML cell lines and PDX models and that FLT3CART also had strong (albeit less complete) activity against FLT3 wild-type AML.…”

Recent Advances in the Development of Anti-FLT3 CAR T-Cell Therapies for Treatment of AML

“… An overview of different anti-FLT3 CAR T-cells developed to date. ( A ) A second-generation CAR with CD28 as co-stimulatory domain [ 30 , 31 , 32 ]. ( B ) A second-generation CAR with 4-1BB as co-stimulatory domain [ 30 , 33 ].…”

“…Karbowski et al present the only study using cynomolgus monkeys to demonstrate tolerability, safety, and dose-dependent efficacy vs. toxicity [ 32 ]. Karbowski et al sought to mimic the human environment as closely as possible to improve prediction of clinical performance and safety of their anti-FLT3 CAR T-cell therapy [ 32 ]. They specifically examined the monkeys for FLT3 expression in tissues that were previously documented to express the FLT3 protein.…”

“…Sommer et al [ 33 ] developed nine anti-FLT3 CARs targeting domain 1–5, and the superior constructs, P3A1 and P3E10, bind to Domains 2 and 4 respectively. Karbowski et al [ 32 ] did not detail the binding location of their scFv.…”