Project Details

CD244-Targeted CAR-T cells to Eradicate Pediatric Acute Myeloid Leukemia Stem Cells

Gabriele Casirati, MD, PhD

Summary

BACKGROUND: Pediatric acute myeloid leukemia (AML) carries a poor prognosis, with a 5-year survival rate of only ~50%. Cellular immunotherapies such as chimeric antigen receptor (CAR) T cells have revolutionized outcomes in other types of leukemias, but their application in AML has been limited by a scarcity of suitable tumor-associated antigens that target leukemia stem cells (LSCs). We here propose to optimize CAR-T cells directed against CD244, a promising and understudied target expressed on LSCs in ~98% of AML cases. GAP: CD244 CAR-T cells have not yet been evaluated beyond preliminary studies. We aim to develop and compare different binders, CAR architectures (hinge regions, intracellular signaling domains), and functional attributes in vitro and in vivo, to identify a lead candidate for clinical translation. HYPOTHESIS: (1) Compare CD244 CAR variants by means of in vitro co-culture assays. (2) Test anti-leukemia efficacy and hematopoietic toxicity of CD244 CAR-T cells in vivo. METHODS: Aim 1a. Through antibody sequencing, we will identify the variable region sequences of novel CD244 binders and clone 2nd gen. CARs into 3rd gen. lentiviral (LV) constructs with different hinge regions and co-stimulatory domains. Aim 1b. Primary T cells will be transduced with these LV vectors to assess CAR-mediated activation, signaling, cytokine secretion, and cytotoxicity against CD244+ and CD244- targets in co-culture. Newly generated CARs will be compared to our validated CD244 CAR, and up to three top-performing constructs will be advanced for in vivo testing. Aim 2a. Immunodeficient NBSGW mice will be humanized by xeno-transplantation of both healthy human CD34+ HSPCs and pediatric patient-derived AML xenografts (PDX), to mimic the co-occurrence of leukemia and healthy hematopoiesis. Candidate CD244 CAR-T cells will be evaluated for their ability to eliminate diverse PDXs while monitoring toxicity on healthy hematopoietic lineages. Analyses will include assessment of residual AML, CAR-T cell expansion/phenotype, and hematopoietic reconstitution in bone marrow and spleen. RESULTS: We have generated CD244 CAR-T cells and demonstrated successful CAR expression and confirmed CAR-mediated T cell activation and high-efficiency killing of CD244+ leukemia cells in vitro. IMPACT: CD244 is widely expressed (>96%) at AML diagnosis and is enriched on LSCs (CD34^+38^-) and relapsed disease. Unlike other AML targets in development (e.g., CD33, CLL-1), CD244’s expression on LSCs suggests a unique opportunity for eradicating residual disease in high-risk pediatric AML patients, potentially before or after bone marrow transplantation. Website Link: www.linkedin.com/in/gabriele-casirati https://genoveselab.dana-farber.org/