Anti-CD19 CAR-T Cells for CD19⁺acute Myeloid Leukemia: A Preclinical Study

Over the last decade, Chimeric Antigen Receptor-modified T (CAR-T) cell therapy has proven a remarkable efficacy in treating refractory B-cell malignancies. CAR-T cells also offer hope in non-B hematological malignancies, albeit with 2 obstacles: the difficulty of antigen selection and the possible resistance to CAR-T cell cytotoxicity due to endogenous tumor cell resistance and/or immunosuppressive microenvironment. In Acute Myeloid Leukemia (AML), the development of CAR-T cells remains difficult, particularly regarding the choice of a target not expressed by normal myeloid cells. However, a subgroup of patients, notably those with a t(8;21) translocation, expresses the CD19 protein on AML cells and could therefore potentially be treated with anti-CD19 CAR-T cells (CART19). Yet, little is known regarding the capacity of CART19 to kill AML cells. The aim of this study was to provide a preclinical proof of principle in vitro and in vivo for the use of CART19 against CD19⁺AML.

For this, we transduced AML-derived Molm-13 cells expressing Green Fluorescence Protein (GFP) and Luciferase (Luc) with a CD19-encoding gammaretroviral vector in order to use them as target cells for in vitro flow cytometry-based cytotoxicity assay and in vivo bioluminescence imaging (BLI). We produced CART19 by lentiviral vector-mediated gene transduction of peripheral lymphocytes from several blood donors. Controls were non-transduced T cells (NT) from the same donors that were cultured in the same conditions than CART19. Immunodeficient NOD/SCID/IL-2 receptor gamma chain^null (NSG) mice were challenged with either 10⁶ Molm-13 or CD19⁺Molm-13 cells. At day 7, they received 5x10⁶ CART19 or NT. Bioluminescence imaging was performed every 7 days after intraperitoneal injection of luciferin. At day 33, a bone marrow aspirate was performed on surviving mice after general anesthesia. Mice were sacrificed in case of high tumor load according to BLI, severe weight loss or paralysis.

CART19 exerted a potent cytotoxic effect in vitro against CD19⁺Molm-13 cells (~60% target cell killing at hour 4 at a 2:1 E:T ratio). Some alloreactivity was observed with NT but did not account for the total cytotoxic effect. This indicated that there was no major intrinsic resistance of AML-derived cells to CAR-T cytotoxicity.

BLI evidenced the capacity of CART19 to kill in vivo Luc⁺CD19⁺Molm-13 cells. By day 22-27, all mice in the control groups (Molm-13-challenged mice receiving CART19 or NT, and CD19⁺Molm-13-challenged mice receiving NT) had to be sacrificed due to leukemia. In these mice, Molm-13 cells but no human CD3⁺ T cells were detected by flow cytometry in the spleen or the bone marrow. In contrast, all CD19⁺Molm-13-challenged mice that had received CART19 were alive and healthy (no sign of graft -versus-host disease) up to day 40 (end of follow-up). At day 33, no tumor burden was detected by BLI, and no Molm-13 cells were found by flow cytometry of bone marrow aspirates whereas CART19 were readily evidenced.

Collectively, these data demonstrate that CART19 can efficiently kill CD19⁺AML cells in vitro and in vivo. With the published report of a case of CD19⁺AML remission after CART19 therapy, these data prompted us to set up a clinical trial based on the injection of academically produced CART19 in patients with relapsed/refractory CD19⁺AML.

Guzman:BridgeMedicines: Research Funding; SeqRX LLC: Current equity holder in private company. Yakoub-Agha:Miltenyi Biomedicine: Honoraria; Novartis: Honoraria; BMS: Honoraria; KITE: Honoraria. Boyer:Janssen: Consultancy, Honoraria, Research Funding; Egle Tx: Consultancy, Honoraria; CSL Behring: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Argenx: Consultancy, Honoraria, Research Funding.