Anticancer DNA vaccine based on human telomerase reverse transcriptase generates a strong and specific T cell immune response

Abstract

Human telomerase reverse transcriptase (hTERT) is overexpressed in more than 85% of human cancers regardless of their cellular origin. As immunological tolerance to hTERT can be overcome not only spontaneously but also by vaccination, it represents a relevant universal tumor associated antigen (TAA). Indeed, hTERT specific cytotoxic T lymphocyte (CTL) precursors are present within the peripheral T-cell repertoire. Consequently, hTERT vaccine represents an attractive candidate for antitumor immunotherapy. Here, an optimized DNA plasmid encoding an inactivated form of hTERT, named INVAC-1, was designed in order to trigger cellular immunity against tumors. Intradermal injection of INVAC-1 followed by electrogene transfer (EGT) in a variety of mouse models elicited broad hTERT specific cellular immune responses including high CD4⁺ Th1 effector and memory CD8⁺ T‑cells. Furthermore, therapeutic INVAC‑1 immunization in a HLA-A2 spontaneous and aggressive mouse sarcoma model slows tumor growth and increases survival rate of 50% of tumor-bearing mice. These results emphasize that INVAC-1 based immunotherapy represents a relevant cancer vaccine candidate.

Keywords: Cancer; DNA vaccines; electrogene transfer; electroporation; hTERT; immunotherapy.

Figure 1.

In vitro characterization of INVAC-1 hTERT protein. (A) Schematic maps and alignments of wild‑type hTERT and INVAC‑1 hTERT proteins. VDD: deletion in catalytic site at position AAs 867‑869. NoLS: Nucleolar Localization Signal. Ubi: ubiquitin, F/R/G: Amino acids flanking the NoLS to Ubi substitution. (B) Expression kinetics of wild‑type hTERT and INVAC‑1 hTERT protein monitored from18 to 96h post‑transfection into HEK293T cells. hTERT protein detection was performed using an anti‑hTERT rabbit monoclonal antibody. NTC: Empty vector backbone as negative control. β‑actin protein detection was used as a loading control assessment. (C) Intracellular localization of wild‑type hTERT and INVAC‑1 hTERT proteins in transfected QT6 cells visualized 24h post‑transfection with an anti‑hTERT rabbit monoclonal antibody and a goat anti‑rabbit secondary antibody‑Alexa Fluor 488® conjugate (green fluorescence). NTC: Empty vector backbone as negative control. NT: Non‑transfected cells. The nuclei were stained with DAPI (blue). The cells were analyzed for both fluorescence wavelengths (merged) upon fluorescence microscopy (x63). (D) Neutralization of INVAC‑1 telomerase catalytic activity. Total cell proteins were extracted from wild‑type hTERT and INVAC‑1 transfected CrFK cells and telomerase activity was assessed by TRAP assay. RTA (sample/positive control ratio) of INVAC‑1 compared to wild‑type hTERT and non-treated (NT) CrFK cells are displayed (n = 3 for 2.1 μg of total protein samples) using absorbance measurements values (OD450/690 nm). Mann–Whitney non-parametric test against non‑treated CrFK cells, **p < 0.01.

Figure 2.

INVAC‑1 induces hTERT specific T‑cell responses in mice. (A) C57BL/6 mice (6 mice per group), BALB/c mice (6‑7 mice per group), HLA‑B7 mice (4‑6 mice per group) and HLA‑A2/DR1 mice (5-6 mice per group) were immunized once. Fourteen days later, an IFNγ ELISpot assay was performed with splenocytes stimulated with a pool of hTERT restricted peptides according to mouse MHC. IFNγ hTERT specific CD8⁺ or CD4⁺ T‑cells/200,000 splenocytes are represented as mean ± SD. Mann–Whitney non-parametric test against mice control, **p < 0.01. (B) HLA‑A2/DR1 mice (3‑5 mice per group) were immunized twice (prime-boost) with INVAC‑1 (D0 and D21). At D31, splenocytes were Ficoll purified and stimulated with a pool of 3 hTERT specific peptides restricted to HLA‑DRB1. Supernatants from stimulated cells were recovered and tested in a cytokine binding assay in order to evaluate the concentration of Th1, Th2 and Th17 cytokines secreted by hTERT specific CD4⁺ T‑cells. Cytokine concentrations in pg/mL are represented as mean ± SD. Mann–Whitney non-parametric test against mice control, *p < 0.05.

Figure 3.

Prime‑boost vaccination enhanced, shortened and broadened hTERT‑specific T‑cell responses. (A) Ten C57BL/6 mice were immunized four times with INVAC-1 (D0, D21, D123 and D144). Peripheral blood was collected before the first immunization D0, and at D7, D14, D21, D33, D40, D118, D132, D139, D154, D159 and D166. PBMCs were Ficoll purified and stimulated with a pool of 4 hTERT specific peptides restricted to the H2‑Kb/Db and analyzed by an IFNγ ELISPOT assay. Black arrows represent days of vaccination. IFNγ hTERT specific CD8⁺ T‑cells/200,000 PBMCs are represented as mean ± SD. Mann–Whitney non-parametric test against D0, *p < 0.05. (B) Seven to 13‑weeks‑old C57BL/6 mice (6 mice per group) were immunized twice (D0 and D21). At D31, splenocytes were Ficoll purified and stimulated with 13 pools of hTERT 15‑mer overlapping peptides (10 peptides/pool) and analyzed by an IFNγ ELISPOT assay. IFNγ hTERT specific T‑cells /200,000 splenocytes are represented as mean ± SD.

Figure 4.

INVAC‑1 induces hTERT specific cytotoxic T‑cells. (A) HLA‑B7 mice (4‑6 mice per group), were immunized twice (D0 and D21). At D31, an ELISpot granzyme B (GrB) assay was performed with splenocytes stimulated with a pool of hTERT HLA‑B7 restricted peptides. GrB hTERT specific CD8⁺ T‑cells/200,000 splenocytes are represented as mean ± SD. Mann–Whitney non-parametric test against mice control, **p < 0.01. (B) C57BL/6 mice (8 mice per group) were immunized twice (D0 and D21). At D31, syngeneic splenocytes, pulsed with individual hTERT peptides restricted to the H2‑Kb/Db (either p660 or p1034) were labeled with CFSE and injected IV to immunized mice. After 15‑18 h, the disappearance of peptide‑pulsed cells in spleens was analyzed by flow cytometry. (C) Percent of killing was presented as mean ± SD. (D) C57BL/6 mice (6 mice per group) were injected s.c. in the right flank with 2 × 10⁵ TC‑1 cells. On day 5 (when tumors are palpable), mice were immunized with INVAC‑1 or left non-treated (NT). Fourteen days later, mice were sacrificed and splenocytes, tumor draining lymph nodes and tumor infiltrated lymphocytes were isolated. An ELISpot IFNγ was performed with H2‑Kb/Db restricted peptides (p429, p660, p1021 and p1034). IFNγ hTERT specific CD8⁺ T-cells/200,000 cells are represented as mean ± SD. Mann–Whitney non-parametric test **p < 0.01.

Figure 5.

INVAC-1 induces antitumor immunity. (A) Murine TERT protein detection into Sarc-T2r cells was performed using an anti‑hTERT rabbit monoclonal antibody. β‑actin protein detection was used as a loading control assessment. (B) Staining of Sarc-T2r cells was achieved with the same anti-hTERT antibody followed by a secondary goat anti‑rabbit PE antibody (orange histogram). Sarc-T2r cells stained with secondary goat anti‑rabbit PE antibody (blue histogram) and unstained Sarc-T2r cells (red histogram) were used as controls. (C) HLA‑A2/DR1 mice (8–9 mice per group) were injected s.c. in the right flank with 2 × 10⁵ Sarc-T2r cells. On days 7 (when tumors are palpable), D14 and D21, mice were immunized with INVAC‑1 or left non-treated (NT). Tumor sizes were measured twice a week. (D) Plots represent tumor volume growth kinetics of individual mice for both groups. (E) Tumor volumes of individual mice on day 34, when the majority of mice were still alive. Tumor volumes are represented as mean ± SD. Mann–Whitney non-parametric test **p < 0 .01. (F) Kaplan–Meier plot of overall survival.