Phase I study of glypican-3-derived peptide vaccine therapy for patients with refractory pediatric solid tumors

Abstract

The carcinoembryonic antigen glypican-3 (GPC3) is a good target of anticancer immunotherapy against pediatric solid tumors expressing GPC3. In this non-randomized, open-label, phase I clinical trial, we analyzed the safety and efficacy of GPC3-peptide vaccination in patients with pediatric solid tumors. Eighteen patients with pediatric solid tumors expressing GPC3 underwent GPC3-peptide vaccination (intradermal injections every 2 weeks), with the primary endpoint being the safety of GPC3-peptide vaccination and the secondary endpoints being immune response, as measured by interferon (IFN)-γ enzyme-linked immunospot assay and Dextramer staining, and the clinical outcomes of tumor response, progression free survival (PFS), and overall survival (OS). Our findings indicated that GPC3 vaccination was well tolerated. We observed disease-control rates [complete response (CR)+partial response+stable disease] of 66.7% after 2 months, and although patients in the progression group unable to induce GPC3-peptide-specific cytotoxic T lymphocytes (CTLs) received poor prognoses, patients in the partial-remission and remission groups or those with hepatoblastoma received good prognoses. The GPC3-peptide vaccine induced a GPC3-specific CTL response in seven patients, with PFS and OS significantly longer in patients with high GPC3-specific CTL frequencies than in those with low frequencies. Furthermore, we established GPC3-peptide-specific CTL clones from a resected-recurrent tumor from one patient, with these cells exhibiting GPC3-peptide-specific cytokine secretion. The results of this trial demonstrated that the GPC3-peptide-specific CTLs induced by the GPC3-peptide vaccine infiltrated tumor tissue, and use of the GPC3-peptide vaccine might prevent the recurrence of pediatric solid tumors, especially hepatoblastomas, after a second CR.

Keywords: CTL; glypican-3 (GPC3); pediatric solid tumors; peptide vaccine; phase I.

Figure 1.

(A) Kaplan-Meier curves for PFS and OS. Patients in the partial-remission and remission groups (not advanced) exhibited longer PFS and OS than those in the progression group (advanced) (p < 0.01 and p < 0.001, respectively). (B) Kaplan-Meier curves for PFS and OS. Hepatoblastoma patients in the partial-remission group exhibited longer PFS and OS than those harboring other pediatric solid tumors. (C) Kaplan-Meier curves for PFS and OS. Patients with GPC3-specfic CTL frequencies ≥10 exhibited longer PFS and OS than those with GPC3-specfic CTL frequencies <10 (p = 0.06 and p < 0.05, respectively).

Figure 2.

Ex vivo IFN-γ ELISPOT assay (A) and ex vivo Dextramer assay (B) were performed using PBMCs obtained from case 6 before the first vaccination and 2 weeks after each vaccination. The raw data are shown. The GPC3 specific spot number indicates the number of GPC3 peptide-specific spot calculated by subtracting the spot number in a well of HIV peptide.

Figure 3.

PBMCs were obtained from each patient before the first vaccination and 2 weeks after each vaccination. Ex vivo IFN-γ ELISPOT assay were performed for all patients. The GPC3 specific spot number indicates the number of GPC3 peptide-specific spot calculated by subtracting the spot number in a well of HIV peptide. The red point indicates the maximum spot number of each patient after vaccinations. The picture is raw data of the maximum spot of each patient.

Figure 4.

Analysis of GPC3_144-152 peptide specific CTL clone. (A) CD8⁺ GPC3-Dextramer⁺ cells in PBMCs of case 6 following peptide stimulation were sorted to a single cell. GPC3_144-152 peptide specific CTL clone was successfully established. (B) Cytotoxicity assay of the CTL clone against GPC3_144-152 or HIV_19-27 peptide pulsed T2 cells. Effector/target (E/T) ratio is 1, 3, 10. (C, D) The GPC3 expression of cancer cell lines were assessed by western blot (C) and quantitative real-time PCR (D). The relative GPC3 mRNA expression (ratio to SK-Hep-1/hGPC3) are shown. (E) The reactivity of CTL clone against cancer cell lines. The CTL clone were co-cultured with cancer cell lines for 20 hour (E/T = 2). The production of IFN-γ were detected by ELISPOT assay. Data are expressed as the mean ± SD. (F) Inhibition of IFN-γ production by anti-HLA class I mAb. GPC3 expressing cancer cell lines used as target cells (E /T = 2). The IFN-γ production of the CTL clone was markedly inhibited by anti-HLA class I mAb as compared with that by IgG2a isotype control. The black bar indicates anti HLA-class I. The white bar indicates isotype control. The antibodies were used at the concentration of 3, 10, 30 μg/ml. Data are expressed as the mean ± SD. (G) The IFN-γ production of the CTL clone against cancer cell lines pretreated with GPC3 siRNA (E/T = 2). The IFN-γ production of the CTL clone was decreased by GPC3 siRNA. Data are expressed as the mean ± SD. nc, negative control.

Figure 5.

Immunological-response assessment in case 2. (A) The raw data of ex vivo IFN-γ ELISPOT assay using PBMCs of case 2. Dextramer assay using PBMCs of case 2 were performed after in vitro peptide stimulation. (B) Ex vivo GPC3-Dextramer staining after vaccination. GPC3-peptide-specific CTL frequency is indicated as the percentage of Dextramer-positive CTLs to CD8-positive cells in the tumor-resected specimen (red circle). (C) Dextramer analysis of the establishment of the GPC3-peptide-specific CTL clones in the tumor-biopsy specimen. (D) IFN-γ ELISPOT assay against SK-Hep-1/vec, SK-Hep-1/hGPC3, and peptide-pulsed T2A24. Effector / target (E / T) ratio = 0.2. (E) Externalized CD107a analysis of the establishment of the GPC3-peptide-specific CTL clones in the tumor-biopsy specimen. T2A24 pulsed with GPC3_298–306 or HIV_583–591 peptide were used as target cells. (F) TNF-α levels in the CTL clone (TIL clone.1) (1.0 × 10⁵ cells/well) after a 24-h co-culture with the indicated target cells (5 × 10⁴ cells/well). Data represent the mean ± standard deviation of triplicate cultures. *p < 0.05.