Targeting CD117 with engineered invariant natural killer T cells (iNKT Cells) as a novel approach for acute myeloid leukemia treatment

Abstract Background: Our research focuses on developing a CAR-based therapy for acute myeloid leukemia (AML) using engineered invariant natural killer T (iNKT) cells that specifically target the CD117 molecule. iNKT cells are a unique subset of innate T cells characterized by an invariant T-cell receptor, that recognizes CD1d often expressed on myeloid malignancies. iNKT cells exhibit significant cytotoxicity against cancer cells that make them potential candidates for adoptive cell therapy. Their ability to minimize or prevent graft-versus-host disease (GVHD) while maintaining robust antitumor activity enhances their appeal as a potential “off-the-shelf” cellular therapy for various hematologic and solid tumors. Aims: To evaluate the therapeutic efficacy of CD117-CAR-iNKT cells in treating AML and to investigate them as a novel option for adoptive cell therapy. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats using gradient centrifugation with Ficoll Pague Plus Media, followed by enrichment of iNKT cells using anti-iNKT microbeads. iNKT cells were stimulated with irradiated allogeneic PBMCs, α-GalCer, and key cytokines (IL2, IL15, IL21) on Days 0 and 10 to promote their expansion. After 14 days, we achieved a 67-80-fold increase in iNKT cell numbers with a purity of 90-98%. These cells were transduced with an MSGV1 gamma-retroviral vector modified to express a CAR sequence comprising a single-chain variable fragment (scFv) that specifically binds to CD117. The retroviral transduction process, enhanced by fibronectin, yielded transfection efficiencies ranging from 72% to 91.2%, depending on the donor source. In vitro cytotoxicity was assessed by co-culturing CD117-CAR-iNKT or CD117-CAR-T cells with luciferase-transduced CD117 leukemia cell lines (Kasumi-1) for 24 hours. In vivo efficacy was evaluated using NSG mice, which were irradiated (250 cGy/mouse) on Day 0 and subsequently engrafted with 1.5 x10e6 luciferase-transduced leukemia cells on Day 4. Three days post-engraftment, mice received either 2.0 x 10e7 CD117-CAR-iNKT cells or untransduced-iNKT cells, supplemented with intraperitoneal IL2 (1,000 units/animal) daily for three days and then every other day for two weeks. Disease progression and tumor burden were monitored through bioluminescent imaging and clinical evaluations. Animals exhibiting severe illness were euthanized, and H&E-stained tissue samples were analyzed by a pathologist to determine the cause of death. Results: CD117-CAR-iNKT cells demonstrated dose-dependent cytotoxic effects comparable to those observed with CD117-CAR-T cells, with improved cytotoxicity compared to untransduced-iNKT cells. Survival analysis of NSG mice (n = 10 per group) treated with 2.0 x 10e7 CD117-CAR-iNKT cells showed significant survival (The median survivals were 50 days for the control group, 52 days for the untransduced iNKT group, and 58 days for the CAR-iNKT group, respectively with a p-value of 0.0256). 40% of the mice in the CAR-iNKT treated group survived 151 days of observation. Of note, no evidence of GVHD was observed across any administered doses of cell therapy. Conclusion: CD117-targeted CAR-iNKT cells show robust and specific cytotoxicity against AML cell lines, including Kasumi-1. The enhanced survival outcomes observed in NSG mice endorse the therapeutic efficacy of CAR-iNKT cells. The absence of GVHD at all administered doses further validate the safety of this novel approach to AML treatment. This study emphasizes the engineered iNKT cells as a potential treatment option for patients with AML, and their future clinical translation.