Endoplasmic reticulum-targeting sequence enhanced the cellular immunity of a tumor-associated antigen L6-based DNA vaccine

Abstract

Cancer vaccine design to effectively eliminate tumors requires triggering strong immune reactions to elicit long-lasting humoral and cellular immunity and DNA vaccines have been demonstrated to be an attractive immunotherapeutic approach. The tumor-associated antigen L6 (TAL6) is overexpressed on the surface of different cancer cells and promotes cancer progression; therefore, it could be a potential target for cancer treatment. We have revealed that a synthetic peptide containing HLA-A2-restricted cytotoxic T lymphocyte (CTL) and B cell epitope can induce cellular and humoral immunity against TAL6-expressing cancer. To enhance the efficacy of immunotherapy, in this report, we designed an endoplasmic reticulum (ER)-targeting sequence (adenovirus E3/19K protein) at the N-terminus of TAL6 to facilitate MHC class I antigen presentation to CD8+ T cells. Transfection of mammalian cells with the plasmid containing TAL6 fused with the ER-targeting sequence (pEKL6) resulted in higher levels of TAL6 antigens in the ER than transfection with the full-length TAL6 (pL6). The plasmid pEKL6 induced both TAL6-specific CTL responses and antibody titers after intramuscular (IM) immunization with electroporation and it elicited higher levels of antigen-specific CTLs in HLA-A2 transgenic mice. Immunization with pEKL6 induced higher levels of protective antitumor immunity against tumor growth than pL6 immunization in thymoma and melanoma tumor animal models. Notably, pEKL6 elicited long-term anti-tumor immunity against the recurrence of cancers. We found that CD4+ T, CD8+ T, and NK cells are all important for the effector mechanisms of pEKL6 immunization. Thus, cancer therapy using an ER-targeting sequence linked to a tumor antigen holds promise for treating tumors by triggering strong immune reactions.

Keywords: DNA vaccine; TAL6; TM4SF1; cytotoxic T lymphocytes.

Conflict of interest statement

None.

AJCR Copyright © 2019.

Figures

Figure 1

DNA vaccine pEKL6 increases the level of tumor-associated antigen L6 in the endoplasmic reticulum. A. Vaccine design: plasmids encode the tumor-associated antigen L6 driven by the CMV promoter. The pEKL6 vaccine additionally encodes the endoplasmic reticulum (ER)-targeting sequence, human adenovirus E3/19K. B. Representative confocal images show the localization of L6 in the ER of mouse melanoma B16F0 cells. The indicated plasmids were transfected into B16F0 cells, and the L6 protein in the ER was monitored using anti-L6 antibody (green), anti-Calnexin antibody (ER marker, red), and DAPI (blue). C. The colocalization rate of L6 and Calnexin was quantified by using Leica LASX software. Data are presented as the means ± SEM of at least three independent experiments. **P

Figure 2

DNA vaccine pEKL6 elicits CTL and ADCC responses in HLA-A2 Tg mice. A. HLA-A2 Tg mice (n = 12 per group) were I.M. immunized twice with the indicated DNA vaccines. Spleen cells from DNA-vaccinated mice were stimulated with irradiated EL4-L6-A2 or EL4-L6 cells (2×104) for 2 hours. After stimulation, the percentage of CD107a+ CD8+ cells was calculated as EL4-L6-A2-stimulated group (%)-EL4-L6-stimulated group (%). B. Spleen cells were harvested from the DNA-immunized mice and stimulated with 10 μg/ml indicated peptides for 48 h. IFN-γ-secreting cells were determined using an IFN-γ ELISPOT assay. Data are presented as the means ± SD. ***P < 0.001. C. CD8+ T cells specific to HLA-A2-restricted TAL6 peptide A2-5 were measured using PE-conjugated A2-5 tetramers. D. TAL6-specific ADCC was determined in a 51Cr release assay. The lysis percentage was calculated as immunized serum (lysis %) - naïve serum (lysis %). The results obtained are expressed as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

DNA vaccine pEKL6 reduces tumor growth and prolongs survival time in WT mice. (A and B) WT mice (n = 5 per group) were I.M. immunized twice by electroporation with the indicated DNA vaccines. EL4-L6 cells (2×105) were injected subcutaneously at 7 days after final immunization. Tumor volume was detected 2-3 times per week (A), and the survival rate was observed (B). (C and D) Mice were injected with B16-L6 cells (2×104) subcutaneously 7 days after the final immunization. The tumor volume was measured (C), and the survival rate was observed (D). (E) The surviving mice in the pEKL6 group from (D) were subcutaneously transplanted tumors again on day 90.

Figure 4

DNA vaccine pEKL6 inhibits lung metastasis of B16-L6 cells in WT mice. WT mice (n = 5 per group) were I.M. immunized twice by electroporation with the indicated DNA vaccines. B16-L6 cells (5×105) were injected intravenously at 7 days after final immunization. Whole lungs from each group were examined after 20 days of cancer cell inoculation.

Figure 5

DNA vaccine pEKL6 elicits anti-tumor effects on tumor animal models through CD4 T, CD8 T, and NK cells. A. WT mice were immunized with the DNA vaccine pEKL6 twice every for 7 days. Five days after the final immunization, antibodies against NK cells, CD4 T cells, and CD8 T cells were I.V. injected (0.5 mg/mouse) to specifically deplete each cell population. At 7 days after the final immunization, B16-L6 cells (2×104) were injected subcutaneously. Tumor size was measured in 2-3 day intervals in each mouse group. B. The survival rate was observed.