Chimeric Antigen Receptor T Cells Targeting NKG2D-Ligands Show Robust Efficacy Against Acute Myeloid Leukemia and T-Cell Acute Lymphoblastic Leukemia

Abstract

CAR T cell approaches to effectively target AML and T-ALL without off-tumor effects on healthy myeloid or T cell compartments respectively are an unmet medical need. NKG2D-ligands are a promising target given their absence on healthy cells and surface expression in a wide range of malignancies. NKG2D-ligand expression in T-ALL has not previously been interrogated, and reports regarding the prevalence and density of NKG2D-ligand expression in AML vary. A first-in-human clinical trial of CART cells targeting NKG2D ligands in patients with AML demonstrated safety and feasibility (Baumeister et al, Cancer Immunology Research 2019). However, at low CAR T cell doses without lymphodepletion clinical efficacy was limited and in this selective patient cohort, NKG2D-ligand expression was only detectable at low levels. To broadly assess the level of NKG2D-ligand expression in AML and T-ALL and evaluate how this correlates with leukemia susceptibility to CAR T cells targeting NKG2D-ligands, we employed NKG2D-fusion protein-based flow-cytometry and RNA sequencing. CAR T cells bearing the full-length native NKG2D-receptor coupled to the CD3ζ cytoplasmic domain were generated using γ-retroviral transduction and then evaluated in functional assays. We show that NKG2D-ligands are expressed in T-ALL cell lines and primary T-ALL and confirm that NKG2D-ligands are frequently expressed at the surface and RNA-level in primary AML, albeit at relatively low levels. We demonstrate that high-expressing AML and T-ALL targets are exquisitely susceptible to in vitro activity of CAR T cells targeting NKG2D-ligands as evidenced by CD107a degranulation, cytotoxicity and production of IFN-γ and TNF-α. Importantly, we show that even low-level ligand expression in primary leukemia targets results in robust NKG2D-CAR activity. Using mass-cytometry, we reveal a distinct phenotypic profile of NKG2D-CAR T cells following incubation with ligand-positive leukemia targets when compared to controls. This includes selective upregulation of ICOS, 41BB, PD-1, LAG-3 and OX40. Given clinical precedent of pharmacologic HDAC inhibition in AML, we next assessed the impact of preincubation with Valproic Acid (VPA) on NKG2D-ligand expression in AML and whether this enhances susceptibility to NKG2D-CART cells in functional assays. We demonstrate that NKG2D-ligand expression can be selectively enhanced in low-expressing AML cell lines and primary AML blasts when AML cells are pre-incubated with 1mM VPA for 24 hours. Such pharmacologic ligand induction significantly augments NKG2D-CAR anti-leukemic activity in functional assays without affecting healthy Peripheral Blood Mononuclear Cells or CAR T cells. Our findings demonstrate that NKG2D-ligands are viable targets for CAR T cell therapy in AML and T-ALL despite low-level cell-surface expression and provide the rationale for the combination of HDAC-inhibitors with NKG2D-CAR T cell therapy as a potential strategy to achieve clinical NKG2D-CAR T cell efficacy in AML. Figure: A) Specific fluorescence intensity of NKG2D-ligand surface expression in Cell lines and Primary Leukemia cells are displayed in comparison B) NKG2D-CAR T cells (CAR) degranulate and produce proinflammatory cytokines during co-culture with AML and T-ALL lines (representative flow plot, left panel), but not to negative controls, whereas T cells transduced with vector lacking the CAR-transgene (Empty) do not. C) NKG2D-CAR T cells but not Empty control T cells display striking killing activity against AML and T-ALL cell lines. D) Preincubation of primary AML blasts with 1mM VPA for 24 hours enhances NKG2D-ligand expression and significantly augments NKG2D-CAR T cell activity, shown here as IFN-γ production during co-culture with 3 independent primary AML samples. Figure Disclosures Dranoff: Novartis: Employment. Ritz:Equillium: Research Funding; Merck: Research Funding; Kite Pharma: Research Funding; Aleta Biotherapeutics: Consultancy; Celgene: Consultancy; Avrobio: Consultancy; LifeVault Bio: Consultancy; Draper Labs: Consultancy; Talaris Therapeutics: Consultancy; TScan Therapeutics: Consultancy.

Engineered Adoptive T-Cell Therapies for Breast Cancer: Current Progress, Challenges, and Potential

“…HER2 intratumoral heterogeneity, for example, is reported in up to 40% of breast cancers and is a potential mechanism for resistance [ 119 ]. Strategies to overcome intratumoral heterogeneity in solid and hematologic malignancies include using epigenetic modulators to increase surface expression of target antigens [ 120 , 121 , 122 ] and targeting multiple antigens expressed throughout the tumor simultaneously [ 120 , 123 , 124 , 125 ]. Few preclinical studies using these approaches to address tumor heterogeneity in breast cancer models exist, and they will be necessary for the success of these therapies in breast cancer.…”

Myeloid Malignancies

“…Targeting FLT3 or CD117 could be an attractive option, again in association with ASCT (Jetani et al 2018;Myburgh et al 2020). Targeting of the Lewis Y antigen and NKG2DL CAR-T cells has also been proposed, but phase 1 trials have shown short response durations, despite reduced toxicity (Ritchie et al 2013;Driouk et al 2019). CAR-T cells targeting CD44v6 mediate potent antitumour effects against AML while sparing normal haematopoietic stem cells (Casucci et al 2013), and a clinical trial is currently ongoing.…”

Overcoming Heterogeneity of Antigen Expression for Effective CAR T Cell Targeting of Cancers

“…They found that decitabine exposure upregulates MUC1 on the surface of pancreatic cancer cells, effectively sensitizing tumor cells to in vitro cytolysis by MUC1-specific CAR T cells (Figure 1A). A more recent example for the use of epigenetic agents as sensitizers for CAR T cell therapy is upregulation of ligands of natural killer (NK)G2D by valproic acid, a potent histone deacetylase (HDAC) inhibitor [55]. NKG2D is an activating receptor expressed on the cell surface of NK cells.…”

“…In a first-in-human clinical trial in patients with acute myeloid leukemia (AML), NKG2D ligand-redirected CAR T cells had limited efficacy, which was attributed to insufficient levels of ligand expression on the surface of leukemic blasts of the patients [62]. The same group now found that NKG2D ligand expression on AML blasts can be selectively enhanced by preincubation with valproic acid, resulting in augmented antileukemic activity in functional in vitro assays [55].…”