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, 8 (50), 86987-87001
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Effective Control of Acute Myeloid Leukaemia and Acute Lymphoblastic Leukaemia Progression by Telomerase Specific Adoptive T-cell Therapy

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Effective Control of Acute Myeloid Leukaemia and Acute Lymphoblastic Leukaemia Progression by Telomerase Specific Adoptive T-cell Therapy

Sara Sandri et al. Oncotarget.

Abstract

Telomerase (TERT) is a ribonucleoprotein enzyme that preserves the molecular organization at the ends of eukaryotic chromosomes. Since TERT deregulation is a common step in leukaemia, treatments targeting telomerase might be useful for the therapy of hematologic malignancies. Despite a large spectrum of potential drugs, their bench-to-bedside translation is quite limited, with only a therapeutic vaccine in the clinic and a telomerase inhibitor at late stage of preclinical validation. We recently demonstrated that the adoptive transfer of T cell transduced with an HLA-A2-restricted T-cell receptor (TCR), which recognize human TERT with high avidity, controls human B-cell chronic lymphocytic leukaemia (B-CLL) progression without severe side-effects in humanized mice. In the present report, we show the ability of our approach to limit the progression of more aggressive leukemic pathologies, such as acute myeloid leukaemia (AML) and B-cell acute lymphoblastic leukaemia (B-ALL). Together, our findings demonstrate that TERT-based adoptive cell therapy is a concrete platform of T cell-mediated immunotherapy for leukaemia treatment.

Keywords: B-cell acute lymphoblastic leukaemia (B-ALL); TCR-redirected T-cells; acute myeloid leukaemia (AML); adoptive cell therapy (ACT); telomerase (TERT).

Conflict of interest statement

CONFLICTS OF INTEREST The authors declare no financial or commercial conflict of interest. Michael I. Nishimura obtained archiving mandate from the NIH.

Figures

Figure 1
Figure 1. Engineered hTERT865-873-specific T-cells selectively recognize HLA-A2+ acute myeloid leukemia in vitro
A. Percentage of leukemic blasts, telomerase expression (WB and lane quantification normalized on β-actin) and enzymatic activity levels in THP1 cell line, PBMCs from AML patients and age-matched HDs (representative cases out of 10 AML patients and 4 HDs are shown). B. AML leukemic cells were recognized in vitro by engineered hTERT865-873-specific T-cells upon 24-hour co-culture, as assayed both by hIFN-γ release assay (upper panel) and flow cytometry cytotoxicity assay (lower panel). Data are mean ± SD of three independent experiments: hTERT865-873 pulsed HLA-A2+ HD PBMCs (n = 3; CTRL+); hHCV1406-1415-pulsed HLA-A2+ HD PBMCs (n = 3; CTRL-); HLA-A2+ HD PBMCs (n = 4); HLA-A2+ PBMCs from AML patients (n = 10); THP1 cell line (n = 3). Statistical analysis was performed with ANOVA test.
Figure 2
Figure 2. Engineered hTERT865-873-specific T-cells selectively recognize HLA-A2+ acute myeloid leukemia in vivo
A. Assessment of hTERT-specific ACT capability to ameliorate mouse survival upon THP1-Luc subcutaneous challenge. Either hTERT865-873- or HCV1406-1415-TCR-engineered T cell were transferred twice, followed by the i.p. administration of IL-2 to test the ability to control tumor growth. B. Kaplan-Meier survival analysis (hTERT n = 8; hHCV n = 6) of a representative experiment. C. NOG mice were then intravenously injected with THP1-Luc cells and treated with three, weekly ACTs of hTERT865-873-specific or control HCV1406-1415-specific T-cells (representative data of 1 out of 2 independent experiments of total n = 12 mice per group). Tumor growth was weekly evaluated by bioluminescence imaging. D. At 27 days from tumor-challenge, leukemic cells spread to spleen and BM was tested by bioluminescence imaging. E. The percentage of infiltrating malignant hCD45+ cells was also evaluated by Flow cytometric analysis and F. by IHC (20X magnification). Statistical analysis was performed with Student's t test.
Figure 3
Figure 3. Engineered hTERT865-873-specific T-cells selectively recognize HLA-A2+ acute lymphoblastic leukemia in vitro
A. Percentage of leukemic blasts, telomerase expression (WB and lane quantification normalized on β-actin) and enzymatic activity levels in ALL-CM cell line, B-cells isolated from PBMCs of B-ALL patients and age-matched HDs (shown some representative cases out of 10 B-ALL patients and 5 HDs). B. Leukemic cells isolated from B-ALL patients were recognized in vitro by engineered hTERT865-873-specific T-cells upon 24-hour co-culture, as assayed both by hIFN-γ release assay (upper panel) and flow cytometry cytotoxicity assay (lower panel). Data are mean ± SD of three independent experiments: hTERT865-873 pulsed HLA-A2+ HD B-cells (n = 3; CTRL+); hHCV1406-1415-pulsed HLA-A2+ HD B-cells (n = 3; CTRL-); HLA-A2+ HD B-cells (n = 5); HLA-A2+ B-cells from B-ALL patients (n = 10); ALL-CM cell line (n = 3). Statistical analysis was performed with ANOVA test.
Figure 4
Figure 4. Engineered hTERT865-873-specific T-cells selectively recognize HLA-A2+ acute myeloid leukemia in vivo
A. Assessment of hTERT-specific ACT capability to ameliorate mouse survival after ALL-CM subcutaneous challenge. Either hTERT865-873- or HCV1406-1415-TCR-engineered T cell were transferred twice, followed by the i.p. administration of IL-2 to test the ability to control tumor growth. B. Kaplan-Meier survival analysis (hTERT n = 7; hHCV n = 6) of a representative experiment. C. Assessment of hTERT-based ACT in controlling ALL-CM cells expansion upon intravenous injection. Mice were treated with three, weekly ACTs of hTERT865-873-specific or control HCV1406-1415-specific T-cells (cumulative graph of 2 independent experiments of total n = 12 mice per group). D. When control mice showed around 60% of circulating malignant cells (red dotted line), tumour cells dissemination into spleen and BM was tested by IHC (20X magnification). Data are mean ± SD of a representative experiment (n = 6 per group). Statistical analysis was performed with Student's t test.
Figure 5
Figure 5. Engineered hTERT865-873-specific T-cells restrain human acute leukemia progression
NOG mice were challenged with PBMCs isolated from two different HLA-A2+ B-ALL patients (B-ALL#1 and B-ALL#2). Mice were treated with 3 weekly ACTs of hTERT865-873- or hHCV1406-1415-specific T-cells, followed by IL-2 administration. A. Tumor progression was calculated evaluating circulating human B-cells. Mice where sacrificed when control mice showed around 80% of circulating malignant cells (red dotted line). B. IHC analysis of hCD20 expression in spleen, BM, liver and kidney (20X magnification). Only for BM (40X magnification), quantification was obtained by flow cytometric analysis. Data are mean ± SD. Statistical analysis was performed with Student's t test (n = 5 per group). C. Survival follow up of the remaining treated mice (B-ALL#1, hTERT n = 7, hHCV n = 5; B-ALL#2 hTERT n = 5, hHCV n = 4). Kaplan-Meier survival analysis: B-ALL#1, hTERT ACT vs. hHCV ACT: p = 0.001; B-ALL#2, hTERT ACT vs. hHCV ACT: p = 0.003.
Figure 6
Figure 6. Phenotype and persistence of transferred T-cells in vivo
A. hTERT865-873-specific T cells showed an effector memory phenotype (CD62LCCR7) before and 4 days after in vivo administration in leukemic mice. Data show representative contour plots. B. Analysis of engineered T cell inhibition markers (LAG3, PD1 and Tim3) pre-injection and 4 days post-injection in leukemic mice. C. Evaluation of circulating TERT-specific T cells at different time points after adoptive transfer in leukemic mice. Representative dot-plots are shown.

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