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 has been reported in a substantial group of patients with AML along with evidence for prognostic significance. However, reports regarding the prevalence and density of NKG2D-ligand expression in AML vary and detailed studies to define whether low level expression is sufficient to trigger NKG2D-ligand directed CART cell responses are lacking. NKG2D ligand expression in T-ALL has not previously been interrogated. Here we report that NKG2D-ligands are expressed in T-ALL cell lines and primary T-ALL. We confirm that NKG2D-ligands are frequently surface expressed in primary AML, albeit at relatively low levels. Utilizing CAR T cells incorporating the natural immune receptor NKG2D as the antigen binding domain, we demonstrate striking in vitro activity of CAR T cells targeting NKG2D-ligands against AML and T-ALL cell lines and show that even low-level ligand expression in primary AML targets results in robust NKG2D-CAR activity. We found that NKG2D-ligand expression can be selectively enhanced in low-expressing AML cell lines and primary AML blasts via pharmacologic HDAC inhibition. Such pharmacologic NKG2D-ligand induction results in enhanced NKG2D-CAR anti-leukemic activity without affecting healthy PBMC, thereby providing 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.
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.
Background Immune-mediated cytopenias (AIC) are challenging complications following allogeneic hematopoietic stem cell transplantation (HSCT). While broad-acting immunosuppressive agents like corticosteroids are often standard of care, several novel therapies which target specific immunological pathways have recently been developed and provide hope for patients with steroid-refractory courses and may limit long-term toxicity. The successful off-label use of the plasma cell depleting anti-CD38 antibody daratumumab was published in several case reports, suggesting efficacy, i.e., in patients with antibody-mediated AIC refractory to previous B cell depletion. We want to share our experience with two children, whom we treated with daratumumab, including one fatal course with uncontrolled disease. Given the absence of substantial data from HSCT registries or prospective trials, we furthermore provide a critical review of the literature on daratumumab treatment of AIC. Case presentations Patient 1 (P1), an 11-year-old girl with lipopolysaccharide-responsive and beige-like anchor protein (LRBA) deficiency who developed immune-mediated thrombocytopenia (AIT) from day +58 after HSCT, showed a complete response to daratumumab after the fourth of six total daratumumab doses. She remains transfusion independent for over a year of follow-up. Previously, her thrombocytopenia was refractory to corticosteroids, rituximab, intravenous immunoglobulins (IVIG), eltrombopag, cyclosporine A, and sirolimus. Patient 2 (P2), a 6-year-old boy with CD40 ligand (CD40L) deficiency, developed both AIT and hemolytic anemia (AIHA) after HSCT on days +58 and +83, respectively, and was also treated with daratumumab after being previously refractory to prednisolone, rituximab, and IVIG. Yet, he did neither respond to daratumumab nor the concomitantly administered methyprednisolone pulse, plasmapheresis, and eculizumab and succumbed due to refractory disease. Conclusion Reviewing the literature on the use of daratumumab for refractory AIC post-HSCT, we consider daratumumab a promising agent for this life-threatening disorder: ten of the twelve patients reached transfusion independency in the literature. However, treatment failures are likely to be underreported. Thus, controlled trials are needed to explore the safety and efficacy of daratumumab in this rare post-HSCT complication.
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