Treatment of acute myeloid leukemia using CD3/CD123 bispecific antibody-engaged γδT cells in a mouse xenograft model

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by dismal clinical outcomes. CD123, theαchain of the IL-3 receptor, is aberrantly overexpressed on AML blasts, representing a promising target for targeted immunotherapy. However, the clinical utility of CD123-targeted immunotherapies has been severely hampered by treatment-associated toxicities that disrupt normal hematopoietic homeostasis. In contrast, γδ T cells, endowed with the unique capacity to recognize antigens in a major histocompatibility complex (MHC)-independent manner, exert minimal cytotoxicity against normal tissues. Upon specific activation and engagement with CD3/CD123 bispecific antibodies (BsAbs), these cells potently and selectively target CD123⁺ AML blasts while mitigating CRS risk, offering a safer and more effective AML therapeutic strategy.

This study aims to investigate a novel immunotherapeutic strategy that employs CD3/CD123 bispecific antibodies (BsAbs) to engage ex vivo expanded γδT cells, thereby enhancing their targeting of CD123⁺ AML blasts and therapeutic efficacy both in vitro and in vivo.

The mononuclear cells were isolated from peripheral blood (PBMCs) and their γδ T cells were subsequently expanded in vitro. The expanded γδ T cells were co-incubated with CD3/CD123 BsAbs to achieve targeted engagement. The anti-AML activity of CD3/CD123 BsAbs-conjugated γδ T cells was evaluated in vitro via cytotoxicity assays and cytokine secretion profiling. For the in vivo assessment, an AML xenograft model was established in NCG mice by inoculation with Molm-13-Luc cells, and the therapeutic efficacy of the CD3/CD123 BsAbs-armed (pre-incubated and washed) γδ T cells was further evaluated in this model.

CD3/CD123 BsAbs- armed γδ T cells exhibited potent and CD123-dependent cytotoxicity against both established CD123⁺ AML cell lines (Molm-13, THP-1) and primary patient-derived AML cells. This cytotoxic effect was accompanied by robust production of the effector cytokines IFN-γand TNF-α, indicating activation of the γδ T cell-mediated immune response. In the NCG mouse xenograft model bearing Molm-13-Luc AML cells, treatment with BsAbs- armed γδ T cells resulted in a significant decrease in leukemic infiltration and a pronounced extension of animal survival when compared with control groups receiving unmodified γδ T cells or BsAbs alone.

The CD3/CD123 BsAbs effectively redirects γδ T cells to selectively target and eliminate AML cells, providing a promising immunotherapeutic strategy for the treatment of AML.