Advancing next-generation CAR-NK therapies targeting CD5 positive T cell malignancies to the clinic

Summary: Patients with relapsed/refractory (R/R) T cell malignancies have poor outcomes and novel therapies are urgently needed. While CAR-T cells have shown remarkable efficacy in patients with B-cell malignancies and multiple myeloma, targeting T-cell malignancies presents unique challenges. One of the major issues is fratricide due to the shared expression of target antigens on both malignant and normal T cells, leading to self-targeting. Additionally, there is a risk of product contamination with malignant T cells during the manufacturing process, and the prolonged life-span of CAR-T cells poses the risk of long-term T-cell aplasia. In contrast, CAR- engineered natural killer (NK) cells offer several advantages. Unlike T-cells, NK cells do not express T cell target antigens such as CD5, eliminating the risk of fratricide. CAR-NK cells also avoid the issue of product contamination with malignant cells, as they can be derived from healthy donors rather than the patient’s own cells. Their shorter life-span reduces the likelihood of prolonged T-cell aplasia. Moreover, CAR-NK cells retain their innate cytotoxicity while also providing tumor specificity against the target antigen through engineering, without the concerns of graft-versus-host disease in the allogeneic setting, cytokine release syndrome or neurotoxicity. Building on our successful first-in-human trial of cord blood (CB) derived CAR19/IL-15 NK cells in patients with relapsed/refractory B-cell malignancies (published in NEJM 2020; Nature Medicine 2024), we now propose a clinical trial targeting CD5 in T cell malignancies. Our engineered CAR-NK cells express an scFv against CD5, IL-15 to enhance persistence, and an inducible caspase-9 (iC9) safety switch (collectively termed iC9/CAR5-28ζ/IL-15 NK cells). A key innovation in this proposal is our novel ex vivo expansion protocol for CAR- NK cells, incorporating IL-12, IL-18, TGF-β and Rapamycin to enhance the metabolic fitness of the cells. Preclinically, iC9/CAR5-28ζ/IL-15 NK cells generated using this novel expansion strategy demonstrated potent activity against T-cell lymphoma models. The clinical protocol has received IRB and FDA (protocol #2021-0526, IND 30087) and is currently enrolling patients. In parallel, we will conduct state-of-the-art correlative studies to comprehensively characterize the infused CAR-NK cells, tracking their persistence, trafficking, and immune modulation in patients. Additionally, we have developed a robust genome-wide CRISPR screening platform to identify novel regulators of CAR-NK cell function and mechanisms of resistance in T cell malignancies. Insights from these studies will inform the development of next-generation CAR-NK cells, optimized to overcome immune evasion strategies and improve patient outcomes. In Aim 1 we will conduct a Phase I/II clinical trial to test the safety and efficacy of iC9/CAR5-28ζ/IL-15 NK cells in patients with CD5+ T cell malignancies. In Aim 2 we will apply comprehensive correlative studies. In Aim 3, we will perform genome-wide CRISPR screens in both our iC9/CAR5-28ζ/IL-15 NK cells and T cell malignancy cell lines to uncover novel mechanisms of resistance and inform the design of next-generation CAR-NK cell therapies.