Harnessing CD33-Targeted CAR T Cells: A Promising Approach for Acute Myeloid Leukemia Therapy

The development of advanced treatments for acute myeloid leukemia (AML) is crucial due to the stagnation in enhancing long-term patient survival. Innovative approaches such as utilizing chimeric antigen receptor (CAR) T cells have been considered for this purpose. However, the challenge lies in identifying a suitable antigen for targeting, as AML cells do not have an ideal target. CD33, which is present in 85-90% of AML cases, is a common target but also resides in normal myeloid cells, posing a risk of toxicity.

Our goal was to develop CAR T cells that target CD33, with the objective of initiating a phase I clinical trial for relapsed AML patients. To mitigate potential risks, an elimination gene was incorporated into the design to facilitate the removal of CAR T cells post-treatment.

We created a CD33-specific CAR using the humanized M-195 antibody's variable chains, along with CD28 and zeta signaling domains, and the IL-12 gene. To ensure the ability to eliminate CAR T cells if required, the EGFRt gene was integrated into the construct, resulting in the EGFRt/HuM195-28z/IL-12 CAR. This construct showed high efficiency in transducing human T cells, as evidenced by flow cytometry using fluorescent cetuximab or CD33.

The engineered T cells produced functional IL-12, which was measured by culturing with peripheral blood mononuclear cells and detecting IFN-g levels. Upon CAR stimulation using the CD33+ AML cell line Molm-13, the CAR T cells showed significant proliferation and produced high levels of IFN-g and IL-2. Furthermore, these cells demonstrated substantial cytotoxicity against Molm-13 in chromium-release assays.

The efficacy of these CAR T cells was further tested in two preclinical mouse models. SCID/Beige mice xenografted with Molm-13 cells showed significantly improved long-term survival when treated with the CAR T cells. Similarly, NSG mice engrafted with patient-derived CD33+ AML cells exhibited a reduction in peripheral CD33+ disease upon treatment.

The findings indicate that the EGFRt/HuM195-28z/IL-12 CAR can effectively target CD33+ AML tumor cells. Ongoing research is focused on validating the elimination mechanism of CAR T cells via EGFRt in vitro and in vivo. The results support the potential use of CD33-targeting CAR T cells in clinical settings for patients with relapsed AML to reduce disease load prior to further therapies such as allogeneic transplantation.