Antitumor effect of new multiple antigen peptide based on HLA-A0201-restricted CTL epitopes of human telomerase reverse transcriptase (hTERT)

Abstract

The development of peptide vaccines aimed at enhancing immune responses against tumor cells is becoming a promising area of research. Human telomerase reverse transcriptase (hTERT) is an ideal universal target for novel immunotherapies against cancers. The aim of this work was to verify whether the multiple antigen peptides (MAP) based on HLA-A0201-restricted CTL epitopes of hTERT could trigger a better and more sustained CTL response and kill multiple types of hTERT-positive tumor cells in vitro and ex vivo. Dendritic cells (DC) pulsed with MAP based on HLA-A0201-restricted CTL epitopes of hTERT (hTERT-540, hTERT-865 and hTERT-572Y) were used to evaluate immune responses against various tumors and were compared to the immune responses resulting from the use of corresponding linear epitopes and a recombinant adenovirus-hTERT vector. A 4-h standard (51) Cr-release assay and an ELISPOT assay were used for both in vitro and ex vivo analyses. Results demonstrated that targeting hTERT with an adenovector was the most effective way to stimulate a CD8(+) T cell response. When compared with linear hTERT epitopes, MAP could trigger stronger hTERT-specific CTL responses against tumor cells expressing hTERT and HLA-A0201. In contrast, the activated CTL could neither kill the hTERT-negative tumor cells, such as U2OS cells, nor kill HLA-A0201 negative cells, such as HepG2 cells. We also found that these peptide-specific CTL could not kill autologous lymphocytes and DC with low telomerase activity. Our results indicate that MAP from hTERT can be exploited for cancer immunotherapy.

Figure 1

Analogue structure of four‐branched peptides of human telomerase reverse transcriptase (hTERT) and phenotypes of target cells. (a) Schematic representation of a multiple antigen peptide containing four peptide monomers: (a) MAP4‐hTERT‐540; (b) MAP4‐hTERT‐865; (c) MAP4‐hTERT‐572Y. (b) Expression of hTERT in target cells: (a) Expression of hTERT mRNA in target cells by RT‐PCR and (b) hTERT protein expression in different target cells as determined by western blot. GAPDH or β‐actin served as reference gene. (1) U2OS; (2) U2OS/hTERT; (3) KATO‐III; (4) SW480; (5) MCF‐7; (6) HepG2; (c) HLA‐A0201 expression in various target cells: SW480, KATO‐III, U2OS, MCF‐7, HepG2, HepG2/HLA‐A0201.

Figure 2

Phenotype of mature dendritic cells (DC). (a) molecular phenotypes in DC generated from PBMC. (b) molecular phenotypes in DC generated from mouse bone marrow.

Figure 3

Specific lysis of KATO‐III, SW480 and MCF‐7 tumor cells by CTL activated by human telomerase reverse transcriptase (hTERT)‐derived peptides in vitro. (a) Specific lysis of CTL induced by adenovector encoding full length hTERT, hTERT‐540‐MAP vaccine or their corresponding linear peptide. (b) Specific lysis of CTL induced by specific lysis of CTL induced by adenovector encoding full length hTERT, hTERT‐865‐MAP vaccine or their corresponding linear peptide. (c) Specific lysis of CTL induced by adenovector encoding full length hTERT, hTERT‐572Y‐MAP vaccine or their corresponding linear peptide. Effector‐to‐target ratios are shown on the x‐axis, and the y‐axis shows percent specific lysis. CTL induced by one nonapeptide from the HIV sequence served as a negative peptide (NP). Results are given as the mean ± SD. ^△Statistically significant values at P < 0.05 using a paired Student's t‐test when tetrameric multiple antigen peptides (MAP) compared with the recombinant adenovirus‐hTERT vector. *Statistically significant values at P < 0.05 using a paired Student's t‐test when tetrameric MAP compared with their corresponding linear peptides.

Figure 4

Specific lysis of KATO‐III, SW480 and MCF‐7 tumor cells by CTL activated by human telomerase reverse transcriptase (hTERT)‐derived peptides ex vivo. (a) Specific lysis of CTL induced by adenovector encoding full length hTERT, hTERT‐540‐MAP vaccine or their corresponding linear peptide. (b) Specific lysis of CTL induced by specific lysis of CTL induced by adenovector encoding full length hTERT, hTERT‐865‐MAP vaccine or their corresponding linear peptide. (c) Specific lysis of CTL induced by adenovector encoding full length hTERT, hTERT‐572Y‐MAP vaccine or their corresponding linear peptide. Effector‐to‐target ratios are shown on the x‐axis, and the y‐axis shows percent specific lysis. CTL induced by one nonapeptide from the HIV sequence served as a negative peptide (NP). Results are given as the mean ± SD. ^△Statistically significant values at P < 0.05 using a paired Student's t‐test when tetrameric multiple antigen peptides (MAP) compared with the recombinant adenovirus‐hTERT vector. *Statistically significant values at P < 0.05 using a paired Student's t‐test when tetrameric MAP compared with their corresponding linear peptides.

Figure 5

hTERT specificity of CTL generated from human telomerase reverse transcriptase (hTERT) vaccines in vitro and ex vivo. (a) Specific lysis of CTL induced by adenovector encoding full length hTERT, multiple antigen peptides (MAP) vaccines or their corresponding linear peptide‐pulsed DC from PBMC against U2OS, U2OS/hTERT in vitro. (b) Specific lysis of CTL induced by adenovector encoding full length hTERT, MAP vaccines or their corresponding linear peptide‐pulsed DC from mouse bone marrow against U2OS, U2OS/hTERT ex vivo. Effector‐to‐target ratios are shown on the x‐axis, whereas the y‐axis shows percent specific lysis. CTL induced by one nonapeptide from the HIV sequence (HIVpol [476–484] [ILLEPVHGV]) served as a negative peptide (NP). Results are given as the mean ± SD. ^△Statistically significant values at P < 0.05 using a paired Student's t‐test when tetrameric MAP is compared with the recombinant adenovirus‐hTERT vector. *Statistically significant values at P < 0.05 using a paired Student's t‐test when tetrameric MAP is compared with their corresponding linear peptides.

Figure 6

HLA‐A0201 restriction of CTL generated from human telomerase reverse transcriptase (hTERT) vaccines in vitro and ex vivo. (a) Specific lysis of CTL induced by adenovector encoding full length hTERT, multiple antigen peptides (MAP) vaccines or their corresponding linear peptide‐pulsed DC from PBMC against HepG2, HepG2/HLA‐A0201 in vitro. (b) Specific lysis of CTL induced by adenovector encoding full length hTERT, MAP vaccines or their corresponding linear peptide‐pulsed DC from mouse bone marrow against HepG2, HepG2/HLA‐A0201 ex vivo. Effector‐to‐target ratios are shown on the x‐axis, whereas the y‐axis shows percent specific lysis. CTL induced by one nonapeptide from the HIV sequence (HIVpol [476–484] [ILLEPVHGV]) served as a negative peptide (NP). Results are given as the mean ± SD. ^△Statistically significant values at P < 0.05 using a paired Student's t‐test when tetrameric MAP is compared with the recombinant adenovirus‐hTERT vector. *Statistically significant values at P < 0.05 using a paired Student's t‐test when tetrameric MAP is compared with their corresponding linear peptides.

Figure 7

The effect of human telomerase reverse transcriptase (hTERT)‐specific CTL on lymphocytes or dendritic cells (DC). Specific lysis of CTL generated from adenovector encoding full length hTERT, multiple antigen peptides (MAP) vaccines or their corresponding linear peptide against autologous lymphocytes and DC in vitro (a) and ex vivo (b). Effector‐to‐target ratios are shown on the x‐axis, whereas the y‐axis shows percent specific lysis. CTL induced using one nonapeptide from the HIV sequence (HIVpol [476–484] [ILLEPVHGV]) served as a negative peptide (NP). Results are given as the mean ± SD.

Figure 8

CD4 T cell depletion reduced inferferon‐gamma (IFN‐γ)‐producing effectors in human telomerase reverse transcriptase (hTERT) peptide immunized mice. Mice vaccinated with MAP4‐hTERT‐540, MAP4‐hTERT‐865 and MAP4‐hTERT‐572Y had increased numbers of IFN‐γ positive cells compared to mice vaccinated with the corresponding linear peptides (hTERT‐540, hTERT‐865 and hTERT‐572Y). However, the number of IFN‐γ‐producing effectors was significantly reduced in CD4‐depleted mice regardless of whether the mice were vaccinated with multiple antigen peptides (MAP) or linear peptides. Phytohemaglutinnin (PHA) was used as a positive control and one nonapeptide from the HIV sequence (HIVpol [476–484] [ILLEPVHGV]) served as a negative control. *Statistically significant values at P < 0.05 using a paired Student's t‐test compared with the corresponding controls. **Statistically significant values at P < 0.01 using a paired Student's t‐test compared with the corresponding controls.