Targeting Telomerase with an HLA Class II-Restricted TCR for Cancer Immunotherapy

Abstract

T cell receptor (TCR)-engineered T cell therapy is a promising cancer treatment approach. Human telomerase reverse transcriptase (hTERT) is overexpressed in the majority of tumors and a potential target for adoptive cell therapy. We isolated a novel hTERT-specific TCR sequence, named Radium-4, from a clinically responding pancreatic cancer patient vaccinated with a long hTERT peptide. Radium-4 TCR-redirected primary CD4⁺ and CD8⁺ T cells demonstrated in vitro efficacy, producing inflammatory cytokines and killing hTERT⁺ melanoma cells in both 2D and 3D settings, as well as malignant, patient-derived ascites cells. Importantly, T cells expressing Radium-4 TCR displayed no toxicity against bone marrow stem cells or mature hematopoietic cells. Notably, Radium-4 TCR⁺ T cells also significantly reduced tumor growth and improved survival in a xenograft mouse model. Since hTERT is a universal cancer antigen, and the very frequently expressed HLA class II molecules presenting the hTERT peptide to this TCR provide a very high (>75%) population coverage, this TCR represents an attractive candidate for immunotherapy of solid tumors.

Keywords: CD4 T cell; MHC class II; T cell receptor; immunotherapy; in vivo model; solid tumor; telomerase.

Graphical abstract

Figure 1

Radium-4 TCR Is Efficiently Expressed, Functional, and Specific (A) Intracellular IFN-γ and TNF-α production in the CD4⁺ patient T cell clone cocultured with HLA-DP04⁺ EBV-LCL, loaded or not with hTERT 611–626 peptide. Representative flow diagram from two independent experiments. (B) Schematic representation of the retroviral construct used for the transduction of Radium-4 TCR in primary T cells. Expression of Radium-4 TCR in primary T cells after retroviral transduction (leftpanels: mock transduced; right panels: TCR transduced) detected by flow cytometry using an anti-CD34 antibody. Data shown are representative flow diagrams from three independent experiments. (C) viSNE representation of phenotypic markers of expanded and transduced Radium-4 TCR T cells. (D) viSNE representation of the expression of extracellular activation markers and immune-checkpoint molecules of Radium-4 TCR T cells upon coincubation with ESTDAB-039 cells, preloaded or not with hTERT 611–626 peptide. (E) Intracellular IFN-γ and TNF-α production in CD4⁺ and CD8⁺ T cells retrovirally transduced with Radium-4 TCR and cocultured with EBV-LCL, loaded or not with hTERT 611–626 peptide. Representative flow diagram from three independent experiments. (F) Summary of intracellular IFN-γ and TNF-α production shown in (C). Bars represent mean ± SD. Statistical validation was realized between conditions in THE presence and absence of peptide. (G) Percentage of cytokine-producing Radium-4 TCR mRNA-electroporated T cells upon coculture with nonloaded APCs or loaded with hTERT 611–626 peptide or hTERT protein. Bars represent mean ± SD of quadruplicates.

Figure 2

Radium-4 TCR Promotes Specific Tumor Lysis (A) Lysis kinetics obtained by BLI assay of effector T cells transduced with Radium-4 TCR (red), Radium-6 (Rad6) TCR (blue), or mock-transduced T cells (black) cocultured with a HLA-DP04⁺ HLA-DR04⁺ EBV-LCL cell line, loaded or not with hTERT 611–626 peptide (phTERT), TGF-βRII frameshift mutated 127–145 peptide (p621), or NY-ESO-1 157–170 peptide (pNY-ESO-1). Representative graphs of two experiments. Data represent mean ± SD of triplicates. Statistics were calculated on the 22-h time point. (B) Corresponding intracellular IFN-γ and TNF-α production of the same effector T cells (CD4⁺ cells) cocultured in a similar fashion as described in (A). Bars represent mean ± SD of triplicates. (C) Measurements of the Annexin V signal restricted to phase/GFP⁺ detected objects of ESTDAB-1000 spheroids in the presence of Rad-4 TCR, Rad-6 TCR, or mock-transduced T cells. Data represent mean ± SD of 36-plicates. Pooled metrics of two independent experiments are shown. (D) Measurements of GFP signal of ESTDAB-1000 spheroids in the presence of Rad-4 TCR, Rad-6 TCR, or mock-transduced T cells. Data represent mean ± SD of 36-plicates. Pooled metrics of two experiments are shown. (E) Representative micrographs of ESTDAB-1000 spheroids in the presence of Radium-4 or Radium-6 TCR-transduced cells or mock T cells. Green and red signals come from ESTDAB-1000 GFP⁺ cells and Annexin V, respectively. Scale bar represents 250 μm.

Figure 3

Radium-4 TCR Recognizes Endogenous Antigen (A) Lysis kinetics obtained by measuring the Annexin V signal of effector T cells transduced with Radium-4 TCR cocultured with patient-derived melanoma cell lines (hTERT⁺/HLA-DP04⁺ or HLA-DP03⁺) or the K562 cell line (hTERT⁺/HLA-DP04⁻). Pooled graphs of two experiments. Data represent mean ± SEM of dodecaplicates. (B) Lysis kinetics obtained by measuring the Annexin V signal of effector T cells transduced with Radium-4 TCR cocultured with spheroids of patient-derived melanoma cell lines (hTERT⁺/HLA-DP04⁺ or HLA-DP03⁺) or the K562 cell line (hTERT⁺/HLA-DP04⁻). Pooled graphs of two experiments. Data represent mean ± SEM of hexaplicates. (C) Representative micrographs of HM8, MM369, and K562 spheroids in the presence of Radium-4 or mock T cells. Red signals come from Annexin V. Scale bar, 250 μm. (D) Annexin V signal after coculture of effector T cell patient ascites cells measured by live-cell imaging. Data represent mean ± SD of octoplicates. Statistics were calculated on the 10-h time point. (E) Representative micrographs of T cells and patient ascites coculture. Red signals come from Annexin V. Scale bar, 250 μm.

Figure 4

Transduced Radium-4 T Cells Control Tumor Load In Vivo upon Intraperitoneal (i.p.) Administration (A) NSG mice were engrafted with GFP/Luc⁺ ESTDAB-1000 tumors i.p., and 3 days after tumor inoculation, mice were randomized and received i.p. injections of mock or Radium-4 TCR-transduced T cells (n = 10 for each group) for a total of 4 injections. (B) ESTDAB-1000 tumor growth curves after mock or Radium-4 TCR-transduced T cell transfer. Data represent means ± SD of two independent experiments pooled. (C) Kaplan-Meier survival curves of mice shown in (D). Survival curves were analyzed with a Mantel-Cox (log-rank) test. (D) Micrographs obtained from In Vivo Imaging System (IVIS) of mice inoculated with ESTDAB-1000 and treated with mock or Radium-4 TCR-transduced T cells. (E) Percentage of GFP⁻/CD3⁺ cells in single-cell suspensions made from tumor or mesenteric lymph node tissues extracted from mice treated with transduced Radium-4 or mock-transduced T cells. Representative flow diagrams from three independent animals. (F) Summary of the proportion of GFP⁻/CD3⁺ cells shown in €. Bars represent mean ± SD. Statistical validation was realized between Radium-4 and mock conditions.

Figure 5

Transduced Radium-4 T Cells Control Tumor Load In Vivo upon Intravenous (i.v.) Administration (A) NSG mice were engrafted with GFP/Luc⁺ ESTDAB-1000 tumors subcutaneously, and 3 days after tumor inoculation, mice were randomized and received i.v. injections of mock-transduced, Radium-4 TCR-transduced, or Radium-6 TCR-transduced T cells or no treatment (n = 10 for each group; 5 for the untreated condition) with a total of 4 injections. (B and C) ESTDAB-1000 tumor growth curves after no treatment or mock, Radium-4 TCR, or Radium-6 TCR T cell transfer. Tumor load was measured by IVIS (B) or caliper (C). Data represent means ± SD of two independent experiments pooled. (D) Corresponding Kaplan-Meier survival curves. Survival curves were analyzed with a Mantel-Cox (log-rank) test. (E) Micrographs obtained from IVIS of mice inoculated with ESTDAB-1000 and left untreated or treated with mock-transduced T cells, Radium-6 TCR-transduced T cells, or Radium-4 TCR-transduced T cells. (F) Percentage of GFP⁻/CD3⁺ cells in single-cell suspensions made from tumor or spleen tissues. Representative flow diagrams from three independent animals.

Figure 6

Redirected Radium-4 T Cells Do Not Recognize Hematopoietic Cells (A) Healthy donor bone marrow progenitor cells were cocultured with autologous T cells, either transfected with Radium-4 TCR mRNA or mock-transfected T cells for 6 h at an E:T ratio of 10:1. The cells were then plated in semisolid methylcellulose progenitor culture for 14 days and scored for the presence of colony-forming unit (CFU)-erythrocyte (E), red; CFU-granulocyte macrophages (GMs), white colonies; and in gray the total number of colonies. Data represent mean ± SD of triplicates. (B) Fold increase in cytokine production (IFN-γ, TNF-α) measured by flow cytometry upon coculture of stably transduced Radium-4 TCR T cells or mock-transduced T cells from three donors (two HLA-DP04⁺ and one HLA-D04⁻) with their respective PBMCs. Data represent mean ± SD of triplicates. (C) Percentage of CD107a⁺-transduced T cells from three donors, as described in (B) cocultured with purified subpopulations of PBMC. PMA-ionomycin and hTERT 611–626 peptide-loaded HLA-DP04⁺ EBV-LCL stimulations were used as a positive control.