Multicenter, phase II clinical trial of cancer vaccination for advanced esophageal cancer with three peptides derived from novel cancer-testis antigens

Abstract

Background: Since a phase I clinical trial using three HLA-A24-binding peptides from TTK protein kinase (TTK), lymphocyte antigen-6 complex locus K (LY6K), and insulin-like growth factor-II mRNA binding protein-3 (IMP3) had been shown to be promising for esophageal squamous cell carcinoma (ESCC), we further performed a multicenter, non-randomized phase II clinical trial.

Patients and methods: Sixty ESCC patients were enrolled to evaluate OS, PFS, immunological response employing ELISPOT and pentamer assays. Each of the three peptides was administered with IFA weekly. All patients received the vaccination without knowing an HLA-A type, and the HLA types were key-opened at the analysis point. Hence, the endpoints were set to evaluate differences between HLA-A*2402-positive (24(+)) and -negative (24(-)) groups.

Results: The OS in the 24 (+) group (n = 35) tended to be better than that in the 24(-) group (n = 25) (MST 4.6 vs. 2.6 month, respectively, p = 0.121), although the difference was not statistically significant. However, the PFS in the 24(+) group was significantly better than that in the 24(-) group (p = 0.032). In the 24(+) group, ELISPOT assay indicated that the LY6K-, TTK-, and IMP3-specific CTL responses were observed after the vaccination in 63%, 45%, and 60% of the 24(+) group, respectively. The patients having LY6K-, TTK-, and IMP3-specific CTL responses revealed the better OS than those not having CTL induction, respectively. The patients showing the CTL induction for multiple peptides have better clinical responses.

Conclusions: The immune response induced by the vaccination could make the prognosis better for advanced ESCC patients.

Trial registration: ClinicalTrials.gov, number NCT00995358.

Figures

Figure 1

Overall survival (OS) and progression free survival (PFS) in all enrolled patients. The OS (A) and PFS (B) were analyzed by the Kaplan-Meier method. OS was measured in days from the 1st vaccination to death and the PFS were measured in days from the1st vaccination to disease progression.

Figure 2

Overall survival and progression free survival of the A24(+) and A24(−) groups. All enrolled patients had received the vaccination without knowing HLA-A status, and the HLA-A genotypes were used for the key-open at analysis point. The OS (A) and PFS (B) were evaluated for each of the HLA-A*2402-positive (24(+)) and -negative (24(−)) groups for the sub-group analysis. MST, median survival time.

Figure 3

Representative immunological monitoring assays detecting antigen-specific CTL response in a patient belonging to the 24(+) group. PBLs obtained from case 2 patient (HLA-A*2402 positive) after the 10th vaccination were cultured in rIL-2 for 14 days with 2 times of LY6K-peptide stimulation. (A) The cultured lymphocytes were subjected to the ELISPOT assay after depletion of CD4-positive cells by magnetic beads. TISI cells were incubated with responder cells in the presence of LY6K peptide or HIV peptide as an irrelevant control, and the spot counts were quantified (B). (C) The cultured lymphocytes were analyzed with HLA-A2402/LY6K-pentamer in the combination with CD8 and CD3 mAbs with flow cytometry. The value of pentamer (+)/CD8(+) among CD3(+) cells was shown. R/S, responder/stimulator.

Figure 4

OS in the A24 (+) group related to immunological monitoring specific to each of the LY6K, TTK, and IMP3 peptides. In the 24(+) group, the in vitro cultured T cells were subjected to ELISPOT assays. The positive CTL responses specific to each of LY6K-, TTK-, and IMP3-peptides after the vaccination were observed in 63%, 45%, and 60% of the patients, respectively. The OS was compared between the patients with positive CTL response (+) and those with negative CTL response (−) for each peptide, the patients with positive CTL response specific to the LY6K-peptide revealed significantly better OS than those without CTL response (A). Similarly, the patients with positive CTL response specific to the TTK-peptide showed significantly better OS than those without CTL response (B). For IMP3, the patients with positive CTL response specific to the peptide tended to have better OS than those without CTL response, although the difference was not statistically significant (C).

Figure 5

OS in the four subgroups of the A24 (+) group classified by the number of the peptides showing the positive CTL responses. The A24(+) patient group was classified into 4 groups according to the number of peptide antigens which induced the CTL responses (0, 1, 2 and 3). The OS tended to be better when the number of the peptides that induced CTL responses was higher.