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

ABSTRACT: BACKGROUND:C-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. METHODS:First, 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. RESULTS:In 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 ? (TNF?), 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. CONCLUSION:Combinatorial treatment with a TNF? 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.

SUBMITTER: Ataca Atilla P

PROVIDER: S-EPMC7497527 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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

Ataca Atilla Pinar P McKenna Mary K MK Tashiro Haruko H Srinivasan Madhuwanti M Mo Feiyan F Watanabe Norihiro N Simons Brian Wesley BW McLean Stevens Alexandra A Redell Michele S MS Heslop Helen E HE Mamonkin Maksim M Brenner Malcolm K MK Atilla Erden E

Journal for immunotherapy of cancer 20200901 2

<h4>Background</h4>C-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.<h4>Methods</h4>First, we optimized the CLL-1 CAR using different spacer, transmembrane and costimulatory sequences. We used a second retroviral vec ...[more]

PMID: 32938629

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Project description:Glioblastoma (GBM) is the most aggressive primary brain tumor in adults and is virtually incurable with conventional therapies. Immunotherapy with T cells expressing GBM-specific chimeric antigen receptors (CAR) is an attractive approach to improve outcomes. Although CAR T cells targeting GBM antigens, such as IL13 receptor subunit α2 (IL13Rα2), HER2, and EGFR variant III (EGFRvIII), have had antitumor activity in preclinical models, early-phase clinical testing has demonstrated limited antiglioma activity. Transgenic expression of IL15 is an appealing strategy to enhance CAR T-cell effector function. We tested this approach in our IL13Rα2-positive glioma model in which limited IL13Rα2-CAR T-cell persistence results in recurrence of antigen-positive gliomas. T cells were genetically modified with retroviral vectors encoding IL13Rα2-CARs or IL15 (IL13Rα2-CAR.IL15 T cells). IL13Rα2-CAR.IL15 T cells recognized glioma cells in an antigen-dependent fashion, had greater proliferative capacity, and produced more cytokines after repeated stimulations in comparison with IL13Rα2-CAR T cells. No autonomous IL13Rα2-CAR.IL15 T-cell proliferation was observed; however, IL15 expression increased IL13Rα2-CAR T-cell viability in the absence of exogenous cytokines or antigen. In vivo, IL13Rα2-CAR.IL15 T cells persisted longer and had greater antiglioma activity than IL13Rα2-CAR T cells, resulting in a survival advantage. Gliomas recurring after 40 days after T-cell injection had downregulated IL13Rα2 expression, indicating that antigen loss variants occur in the setting of improved T-cell persistence. Thus, CAR T cells for GBM should not only be genetically modified to improve their proliferation and persistence, but also to target multiple antigens.Summary: Glioblastoma responds imperfectly to immunotherapy. Transgenic expression of IL15 in T cells expressing CARs improved their proliferative capacity, persistence, and cytokine production. The emergence of antigen loss variants highlights the need to target multiple tumor antigens. Cancer Immunol Res; 5(7); 571-81. ©2017 AACR.

Project description:BackgroundThe therapeutic application of T cells endowing with chimeric antigen receptors (CARs) is faced with "on-target, off-tumor" toxicity against solid tumors, particularly in the treatment of the pancreatic cancer. To our best knowledge, the pancreatic cancer cell line AsPC-1 often highly expressed some distinct tumor-associated antigens, such as carcino-embryonic antigen (CEA) and mesothelin (MSLN). Therefore, in this research, we have characterized dual-receptor CAR-modified T cells (dCAR-T) that exert effective and safe cytotoxicity against AsPC-1 cells.MethodsBased on the dual signaling pathway of wild T cells, we designed a novel dCAR diagram specific for CEA and MSLN, which achieved comparable activity relative to that of conventional CAR-T cells (CEA-CAR T or MSLN-CAR T). In this dCAR, a tandem construct containing two physically separate structures, CEA-CD3ζ and MSLN-4/1BB signaling domains were effectively controlled with tumor antigens CEA and MSLN, respectively. Finally, the activity of dCAR-T cells has been verified via in vitro and in vivo experiments.ResultsIn the presence of cognate tumor cells (AsPC-1) expressing both CEA and MSLN, dCAR-T cells exerted high anti-tumor activity relative to that of other single-receptor CAR-T cells bearing only one signaling pathway (e.g., Cζ-CAR and MBB-CAR). In a xenograft model, dCAR-T cells significantly inhibited the growth of AsPC-1 cells yet no effect on the growth of non-cognate tumor cells. Furthermore, the released cytokines and T cell persistence in mice were comparable with that of conventional CAR-T cells, obtaining specific and controllable cytotoxicity.ConclusionsA novel type of CAR-T cells, termed dCAR-T, was designed with specific activities, that is, significant cytotoxicity for two antigen-positive tumor cells yet no cytotoxicity for single antigen-positive tumor cells. Dual-targeted CAR-T cells can be precisely localized at the tumor site and can exert high cytotoxicity against tumor cells, alleviating "on-target, off-tumor" toxicity and enabling accurate application of CAR-T cell therapy.

Dataset Information

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

Publications

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

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