Modulating TNFα activity allows transgenic IL15-Expressing CLL-1 CAR T cells to safely eliminate acute myeloid leukemia

Abstract

BackgroundC-type lectin-like molecule 1 (CLL-1) is highly expressed in acute myeloid leukemia (AML) but is absent in primitive hematopoietic progenitors, making it an attractive target for a chimeric antigen receptor (CAR) T-cell therapy. Here, we optimized our CLL-1 CAR for anti-leukemic activity in mouse xenograft models of aggressive AML.MethodsFirst, we optimized the CLL-1 CAR using different spacer, transmembrane and costimulatory sequences. We used a second retroviral vector to coexpress transgenic IL15. We measured the effects of each construct on T cell phenotype and sequential (recursive) co culture assays with tumor cell targets to determine the durability of the anti tumor activity by flow cytometry. We administered CAR T cells to mice engrafted with patient derived xenografts (PDX) and AML cell line and determined anti tumor activity by bioluminescence imaging and weekly bleeding, measured serum cytokines by multiplex analysis. After euthanasia, we examined formalin-fixed/paraffin embedded sections. Unpaired two-tailed Student’s t-tests were used and values of p<0.05 were considered significant. Survival was calculated using Mantel-Cox log-rank test.ResultsIn vitro, CLL-1 CAR T cells with interleukin-15 (IL15) were less terminally differentiated (p<0.0001) and had superior expansion compared with CD28z-CD8 CAR T cells without IL15 (p<0.001). In both AML PDX and AML cell line animal models, CLL-1 CAR T coexpressing transgenic IL15 initially expanded better than CD28z-CD8 CAR T without IL15 (p<0.0001), but produced severe acute toxicity associated with high level production of human tumor necrosis factor α (TNFα), IL15 and IL2. Histopathology showed marked inflammatory changes with tissue damage in lung and liver. This acute toxicity could be managed by two strategies, individually or in combination. The excessive TNF alpha secretion could be blocked with anti-TNF alpha antibody, while excessive T cell expansion could be arrested by activation of an inducible caspase nine safety switch by administration of dimerizing drug. Both strategies successfully prolonged tumor-free survival.ConclusionCombinatorial treatment with a TNFα blocking antibody and subsequent activation of the caspase-9 control switch increased the expansion, survival and antileukemic potency of CLL-1 CAR T-cells expressing transgenic IL15 while avoiding the toxicities associated with excessive cytokine production and long-term accumulation of activated T-cells.

Current Limitations and Perspectives of Chimeric Antigen Receptor-T-Cells in Acute Myeloid Leukemia

“…IDO catalyzes the conversion of tryptophan into immunosuppressive metabolites [ 142 ] that inhibit the expansion of CAR-T-cells in response to interleukins such as IL-7 or IL-15. Approaches to attenuate the deleterious effects of IDO include engineering strategies to equip CAR-T-cells with IL-secreting properties [ 143 , 144 ] or combining CAR-T-cell therapy with the tyrosine kinase inhibitor sorafenib that increases IL-15 production in FLT3-ITD+ AML [ 145 ].…”

Engineered Cytokine Signaling to Improve CAR T Cell Effector Function

“…Constitutive expression of IL-15 improved the antitumor activity of CAR T cells specific for CD19, GPC-3, CLL-1, GD2 and IL-13Rα2 ( 75 – 79 ), likely due to a combination of greater expansion and persistence. Constitutive expression of IL-15 by first generation CD19-CAR T cells improved their in vivo antitumor activity and allowed cells to persist in mice for up to 110 days after tumor challenge ( 71 ).…”

Advances in CAR-T Cell Genetic Engineering Strategies to Overcome Hurdles in Solid Tumors Treatment

“…Therefore, transgenic expression of IL-15 seemed like an appealing strategy to enhance CAR-T cell effector function, by enhancing proliferation and persistence of CAR-T cells in the TME and has been used in preclinical models of acute myeloid leukemia (AML), melanoma, glioblastoma or neuroblastoma ( 261 – 265 , 273 ). In preclinical models of AML, CLL1-directed CAR-T cells with transgenic expression of IL-15 showed increased expansion, survival and antileukemic potency.…”

“…In preclinical models of AML, CLL1-directed CAR-T cells with transgenic expression of IL-15 showed increased expansion, survival and antileukemic potency. Unfortunately, co-expression of IL-15 was associated with lethal cytokine release syndrome (CRS), a fatal adverse effect that could be prevented with anti-TNF-antibodies pretreatment and depletion of IL-15 secreting CARs by the inducible caspase-9 (iCas-9) suicide switch ( 261 ).…”