Vaccination with peptides derived from cancer-testis antigens in combination with CpG-7909 elicits strong specific CD8+ T cell response in patients with metastatic esophageal squamous cell carcinoma

Abstract

Potent helper action is necessary for peptide-based vaccines to efficiently induce antitumor immune responses against advanced cancer. A phase I trial for advanced esophageal squamous cell carcinoma was carried out for patients with HLA-A*2402 using epitope peptides derived from novel cancer-testis antigens, LY6K and TTK, in combination with CpG-7909 (NCT00669292). This study investigated the feasibility and the toxicity as well as induction of tumor antigen-specific immune responses. Nine patients were vaccinated on days 1, 8, 15, and 22 of each 28-day treatment cycle with peptide LY6K-177, peptide TTK-567, and CpG-7909 (level-1; 0, level-2; 0.02, level-3; 0.1 mg/kg) and all were tolerated by this treatment. LY6K-specific T cell responses in PBMCs were detected in two of the three patients in each level. In particular, two patients in level-2/3 showed potent LY6K-specific T cell responses. In contrast, only two patients in level-2/3 showed TTK-567-specific T cell responses. The frequency of LY6K-177 or TTK-567-specific CD8+ T cells increased in patients in level-2/3 (with CpG). The vaccination with peptides and CpG-7909 increased and activated both plasmacytoid dendritic cells and natural killer cells, and increased the serum level of α-interferon. There were no complete response (CR) and partial response (PR), however, one of three patients in level-1, and four of six patients in level-2/3 showed stable disease (SD). In conclusion, vaccination with LY6K-177 and TTK-567 in combination with CpG-7909 successfully elicited antigen-specific CD8+ T cell responses and enhanced the innate immunity of patients with advanced esophageal squamous cell carcinoma. This vaccine protocol is therefore recommended to undergo further phase II trials.

Figure 1

(A) Vaccine‐generated LY6K‐177‐specific T cell response. LY6K‐specific T cell responses were assessed by enzyme‐linked immunosorbent spot assay. The PBMCs were cultured with LY6K‐177 peptide for 2 weeks. On day 7, cells were restimulated with LY6K‐177 peptide. The harvested cells were used as responder cells and LY6K‐177‐pulsed TISI cells were used as stimulator cells. HLA‐A*2402‐restricted epitope peptide derived from HIV‐Env protein was used as the negative control peptide. Spots were captured and analyzed using ImmunoSPOT 4S. Responder cells were mixed with stimulator cells at different ratio, and incubated for 24 h. The X‐axis of the figure indicates the responder to stimulator (R/S) ratio. The Y‐axis indicates the number of specific spots. (B) Vaccine‐generated TTK‐567‐specific T cell response. TTK‐specific T cell responses were assessed by enzyme‐linked immunosorbent spot assay. The PBMCs were cultured with TTK‐567 peptide for 2 weeks. The cells were restimulated with TTK‐567 peptide on day 7. The harvested cells were used as responder cells and TTK‐567‐pulsed TISI cells were used as stimulator cells. HLA‐A*2402‐restricted epitope peptide derived from HIV‐Env protein was used as the negative control peptide. Responder cells were mixed with stimulator cells at different ratios, and incubated for 24 h. Spots were captured and analyzed using ImmunoSPOT 4S. The X‐axis of the figure indicates the R/S ratio. The Y‐axis indicates the number of specific spots.

Figure 2

Frequencies of LY6K/TTK‐specific CD8+ T cells in patients with advanced esophageal squamous cell carcinoma, evaluated by HLA‐A*2402/LY6K‐177 (A) or TTK‐567 (C) pentamer staining. The PBMCs were incubated with phycoerythrin‐labeled HLA‐A*2402 presenting LY6K/TTK for 10 min. We added 7‐AAD, and cells were incubated with Fluorotag and FITC‐conjugated anti‐human CD8 mAb, then analyzed using a FACSCalibur. (B) The representative dot plot data of pentamer staining derived from two positive cases, case 6 and 8.

Figure 3

Effect of vaccination on the population of PBMCs in patients with advanced esophageal squamous cell carcinoma. The phenotypical analysis of PBMCs was carried out using a FACSCalibur with CellQuest software before and after vaccination. Data are expressed as the percent change of the positive cell populations. A percent change of 1.5 times was defined as increase. (A) BDCA2+. (B) CD80+/BDCA2+. (C) CD86+/BDCA2+. (D) CD56+. (E) CD69+/CD56+.