NK Cell Engineering to Target Intracellular Antigens in AML - Summary: Patients with relapsed/refractory (R/R) acute myeloid leukemia (AML) and high-risk myelodysplastic syndromes (MDS) have limited treatment options and poor survival after relapse. While CAR T cell therapies have transformed the treatment of B-cell cancers, their application in AML has been hindered by the absence of safe, leukemia-specific surface antigens and the high toxicity profile of T cell therapies. Additionally, heterogeneous antigen expression on blasts promotes resistance and escape from therapies targeting a single surface marker. AML blasts, however, are susceptible to natural killer (NK) cell killing as they express stress ligands recognized by NK cell activating receptors. Thus, to overcome the challenges of antigen escape, manufacturing logistics, and toxicity, we have developed a first-in-class, cord blood (CB)-derived, off-the-shelf NK cell therapy engineered to express a T cell receptor (TCR) targeting PRAME, a cancer-testis antigen expressed in most AML and high-risk MDS cases but absent from normal hematopoietic stem cells. These PRAME-TCR/IL-15 NK cells co-express the full CD3 complex for TCR signaling, a costimulatory domain to enhance activation, and secreted IL-15 to improve persistence and metabolic fitness. This platform combines the intrinsic antitumor properties of NK cells with the antigen specificity of T cells. This is the first clinical trial to test a TCRengineered NK cell therapy targeting an intracellular antigen and builds on our prior success with CB-derived CAR NK cells s (NEJM 2020; Nature Medicine 2024). The protocol has received IRB and FDA approval (Protocol 2024-0196, IND 30518), and enrollment is ongoing. We have established a GMP-grade cryopreserved biobank of >30 billion PRAME-TCR/IL-15 NK cells, enabling immediate bedside delivery. Our central hypothesis is that that PRAME-TCR/IL-15 NK cells will show clinical activity with reduced toxicity in AML and that paired immune profiling and CRISPR-based functional genomics will reveal key regulators of therapeutic response and resistance. In Aim 1 we will conduct a Phase I/ II clinical trial to evaluate the safety and efficacy of PRAME-TCR/IL-15 NK cells NK cells in patients with R/R AML and high-risk MDS. In Aim 2 we will perform innovative correlative studies to track the fate and functional dynamics of the adoptively infused engineered TCR-NK cells in vivo, using advanced single-cell proteomic and transcriptomic approaches. In Aim 3, we will apply genome-wide CRISPR knockout screens to identify tumor-intrinsic and NK cell intrinsic regulators of therapy response and resistance, followed by in vitro and in vivo validation of top targets. This study represents a new class of immunotherapy, combining the innate safety, antitumor cytotoxicity and scalability of NK cells with the precision of TCR targeting. If successful, this platform could be extended to other PRAME+ cancers and reshape the landscape of allogeneic immunotherapy. Animal models are required to assess the in vivo persistence, trafficking, antitumor activity and safety of PRAME TCR-engineered NK cells in the physiologic context of disseminated AML. These dynamic interactions between tumor burden, tissue distribution, NK cell expansion and toxicity cannot be adequately modeled in vitro and are necessary to support translation of this therapeutic approach.
