Abstract LB102: Off-the-shelf cord blood FLT3 CAR-NK cells for immunotherapy of acute myeloid leukemia
Authors: Mansour Anthony G. , Teng Kun-Yu , Li Zhiyao , Zu Zheng , Chen Hanyu , Ali Aliya , Zhang Jianying , Lu Ting , Ma Shoubao , Caligiuri Michael A. , Yu Jianhua 
Published: June 2022

Abstract
Despite the unprecedented clinical success of autologous chimeric antigen receptor (CAR) T cell therapy in B cell leukemia and lymphoma patients, CAR T cell therapy for acute myeloid leukemia (AML) patients has lagged, in part due to the time required for autologous CAR T cell preparation in the face of the rapid relapse of AML following remission, and the possibility of on-target off-tumor hematopoietic toxicity. In this study we investigated the functional activity of an allogeneic, off-the-shelf FLT3 CAR natural killer (NK) cells expressing soluble (s) IL-15 (FLT3 CAR_s15 NK) to treat FLT3+ AML. Cord blood NK cells were first transduced with FLT3 CAR intracellular signaling constructs containing either CD28/CD3ζ or 2B4/CD3ζ. Each displayed similar cytotoxicity against FLT3+ AML in vitro (p &lt; 0.05) and in vivo (p &lt; 0.01) when compared to mock transduced NK cells. Cord blood NK cells transduced with either soluble (s) IL-15 (s15 NK cells) or membrane (m) bound IL-15 showed comparable extended survival in vivo and superior to NK cells transduced with sIL-15/IL-15Rα (p &lt; 0.01) or mIL-15/IL-15Rα (p &lt; 0.01). However, only those NK cells transduced with soluble IL-15 were able to activate neighboring non-transduced (NT) NK cells and T cells in a paracrine fashion. The FLT3 CAR_s15 NK cells were expanded ex-vivo to over 1500-fold in 16 days with ~50% of the NK cells expressing both the FLT3 CAR and sIL-15. These FLT3 CAR_s15 NK cells were then subjected to a cytotoxicity assay against the FLT3+ AML cell line (MOLM-13) and were 10 times more potent than NT NK cells (p &lt; 0.01) and 1.7 times more potent than s15 NK cells (p &lt; 0.05). Likewise, when co-cultured with the FLT3+ AML cell line, FLT3 CAR_s15 NK cells produced a greater amount of IFN-γ compared to NT NK cells (p &lt; 0.01) or compared to s15 NK cells (p &lt; 0.05). Furthermore, following cryopreservation, our thawed FLT3 CAR_s15 NK cells demonstrated high recovery (~90%) and viability (&gt; 85%), maintaining their FLT3 CAR expression at ~50% with a phenotype and cytotoxicity that was nearly equivalent to fresh FLT3 CAR_s15 NK cells. Armed with these data we assessed the efficacy of our allogeneic, off-the-shelf FLT3 CAR_s15 NK cells in vivo using immunodeficient mice engrafted with the MOLM-13 FLT3+ cell line. Repeated infusions of FLT3 CAR_s15 NK cells provided an improved median survival compared to placebo group (36 vs 24 days; p &lt; 0.001) or compared to identical infusions of s15 NK cells (36 vs 31 days; p &lt; 0.05) with similar results obtained in a patient-derived xenograft model. Finally, to assess toxicity against normal peripheral blood mononuclear cells (PBMC), including FLT3+ dendritic cells, and against hematopoietic stem cells (HSCs) we performed in vitro and in vivo studies, respectively, using our FLT3 CAR_s15 NK cells. We did not observe an untoward cytotoxicity of PBMC in vitro when compared to NT or s15 NK cells and no disruption of HSC differentiation in vivo when compared to placebo group. In summary, our in vitro and in vivo studies with allogeneic off-the-shelf cord blood derived FLT3 CAR_s15 NK cells demonstrated enhanced cytotoxicity and IFN-γ secretion against FLT3+ AML in vitro and enhanced the survival of mice engrafted with FLT3+ AML without any evidence of PBMC or HSC toxicity. We believe these data collectively support a rationale for investigating this novel form of allogeneic, off-the-shelf FLT3 CAR_s15 NK cell therapy in patients stricken with FLT3+ AML.
Citation Format: Anthony G. Mansour, Kun-Yu Teng, Zhiyao Li, Zheng Zu, Hanyu Chen, Aliya Ali, Jianying Zhang, Ting Lu, Shoubao Ma, Michael A. Caligiuri, Jianhua Yu. Off-the-shelf cord blood FLT3 CAR-NK cells for immunotherapy of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB102.