Purpose: The carcinoembryonic antigen glypican-3 (GPC3) is an ideal target of anticancer immunotherapy against hepatocellular carcinoma (HCC). In this nonrandomized, open-label, phase I clinical trial, we analyzed the safety and efficacy of GPC3 peptide vaccination in patients with advanced HCC.

Experimental Design: Thirty-three patients with advanced HCC underwent GPC3 peptide vaccination (intradermal injections on days 1, 15, and 29 with dose escalation). The primary endpoint was the safety of GPC3 peptide vaccination. The secondary endpoints were immune response, as measured by IFN-γ ELISPOT assay, and the clinical outcomes tumor response, time to tumor progression, and overall survival (OS).

Results: GPC3 vaccination was well-tolerated. One patient showed a partial response, and 19 patients showed stable disease 2 months after initiation of treatment. Four of the 19 patients with stable disease had tumor necrosis or regression that did not meet the criteria for a partial response. Levels of the tumor markers α-fetoprotein and/or des-γ-carboxy prothrombin temporarily decreased in nine patients. The GPC3 peptide vaccine induced a GPC3-specific CTL response in 30 patients. Furthermore, GPC3-specific CTL frequency after vaccination correlated with OS. OS was significantly longer in patients with high GPC3-specific CTL frequencies (N = 15) than in those with low frequencies (N = 18; P = 0.033).

Conclusions: GPC3-derived peptide vaccination was well-tolerated, and measurable immune responses and antitumor efficacy were noted. This is the first study to show that peptide-specific CTL frequency can be a predictive marker of OS in patients with HCC receiving peptide vaccination. Clin Cancer Res; 18(13); 3686–96. ©2012 AACR.

This article is featured in Highlights of This Issue, p. 3493

Translational Relevance

A cancer vaccine that induces CTLs to tumor-associated antigens is a potentially attractive option for hepatocellular carcinoma (HCC). However, thus far, immunotherapy using tumor antigen–derived peptides has not showed a correlation between immunologic responses and antitumor efficacy in clinical trials in patients with advanced HCC. Glypican-3 (GPC3) is an ideal target for anticancer immunotherapy against HCC because it is specifically overexpressed in HCC and correlates with poor prognosis.

In a phase I clinical study, we investigated the safety and antitumor effects of, and immunologic response to, a GPC3-derived peptide vaccine. Our results show that GPC3 peptide–specific CTLs appeared in peripheral blood and that many CD8-positive T cells infiltrated tumors after GPC3 peptide vaccination.

This is the first study to show that peptide-specific CTL frequency was correlated with overall survival in patients with HCC receiving peptide vaccination. These observations suggest that GPC3-derived peptide vaccines could be a novel therapy for patients with HCC.

While primary liver cancer, which predominantly consists of hepatocellular carcinoma (HCC), is the sixth most common cancer worldwide, it has a very poor prognosis, which makes it the third leading cause of cancer mortality (1). One of the major reasons for the poor prognosis of HCC is the limited availability of treatment options for advanced disease. The molecular-targeted agent sorafenib was recently proven to prolong overall survival (OS) in patients with advanced HCC and has become the standard drug for first-line systemic treatment (2, 3). However, according to Response Evaluation Criteria in Solid Tumors (RECIST), the response rate for sorafenib is quite low, and the incidence of adverse drug reactions is high, especially in elderly patients (4). Moreover, no second-line treatment has been established for patients when sorafenib treatment has failed. Therefore, new treatment modalities are urgently required to prolong survival in patients with advanced HCC while minimizing the risk of adverse reactions.

Immunotherapy is a potentially attractive option for HCC. Many tumor antigens identified in HCC are potential antigens for peptide vaccines (5, 6). However, thus far, immunotherapy using tumor antigen–derived peptides has not showed adequate antitumor efficacy in clinical trials in patients with advanced HCC (7–9). The carcinoembryonic antigen glypican-3 (GPC3) is an ideal target for anticancer immunotherapy against HCC because it is specifically overexpressed in HCC (72%–81%) and correlates with a poor prognosis (10–14). We identified HLA-A*24:02–restricted GPC3298–306 (EYILSLEEL) and HLA-A*02:01–restricted GPC3144–152 (FVGEFFTDV) as peptides that can induce GPC3-reactive CTLs without inducing autoimmunity (15, 16). Moreover, by conducting a binding assay, we confirmed that HLA-A*02:01–restricted GPC3144–152 (FVGEFFTDV) peptide can bind to HLA-A*02:06 and HLA-A*02:07. HLA-A24 is the most common HLA class I allele in the Japanese population, and 60% of Japanese individuals (95% of whom have an A*24:02 genotype), 20% of Caucasians, and 12% of Africans are positive for HLA-A24 (17, 18). HLA-A2 is also expressed in Japanese (40%) and other ethnic populations, with an estimated frequency of 50% in Caucasians (17, 19). In a preclinical study using a mouse model, we developed an optimal schedule for human clinical trials of a GPC3-derived peptide vaccine (20). On the basis of these results, we conducted a phase I clinical trial of this GPC3-derived peptide vaccine in patients with advanced HCC. We previously reported that several GPC3144–152 peptide-specific CTL clones were established from peripheral blood mononuclear cells (PBMC) of patients vaccinated with HLA-A2–restricted GPC3144–152 peptide in this trial (21). We recently completed this phase I clinical trial of the GPC3-derived peptide vaccine. We evaluated the vaccine's safety, tolerability, recommended phase II dose, and immunologic and clinical responses in this trial.

Patient eligibility

This phase I trial was approved by the Ethics Committee of the National Cancer Center and was carried out from February, 2007, to November, 2009. Patients with advanced or metastatic HCC were enrolled after providing written, informed consent. The following eligibility criteria were used: diagnosis of HCC on the basis of imaging modalities or histologic examinations; no expectation of response to other therapies; an Eastern Cooperative Oncology Group performance status of 0–1; age between 20 and 80 years; no prior therapy within 4 weeks; life expectancy ≥3 months; HLA-A24- or HLA-A2–positive status, as determined using commercially available genomic DNA typing tests (Mitsubishi Chemical Medience); Child–Pugh liver function class A and B; and adequate organ function (white blood cell count ≥3,000/μL, hemoglobin ≥8.0 g/dL, platelets ≥50,000/μL, total bilirubin ≤3.0 mg/dL, aspartate aminotransferase ≤200 IU/L, alanine aminotransferase ≤200 IU/L, and serum creatinine ≤1.5 mg/dL). The following exclusion criteria were applied: massive ascites; known brain metastasis; pregnancy or lactation; known history of HIV infection; clinically serious infection; severe cardiac insufficiency; other active malignancy; history of organ allograft; immunodeficiency or history of splenectomy; concurrent treatment with steroids or immunosuppressive agents; and unsuitability for the trial, based on clinical judgment.

Study design and endpoints

This study was a nonrandomized, open-label, phase I clinical trial with dose escalation of the GPC3 peptides in patients with advanced HCC. HLA-A*24:02–restricted GPC3298–306 peptide (EYILSLEEL; American Peptide Company) was used in HLA-A24–positive patients and HLA-A*02:01–restricted GPC3144-152 peptide (FVGEFFTDV; American Peptide Company) in HLA-A2–positive patients. Peptides were administered in liquid form, emulsified with incomplete Freund's adjuvant (IFA; Montanide ISA-51VG, SEPPIC), by intradermal injection on days 1, 15, and 29. The peptides and IFA were synthesized according to Good Manufacturing Practice guidelines. Administration of 5 incremental doses of peptide (0.3, 1.0, 3.0, 10, and 30 mg/body) was planned. We planned administer each dose to 6 patients, including at least each 2 patients given HLA-A2 or A24-restricted peptide. The primary endpoint was the safety of peptide vaccination. The secondary endpoints were immunologic responses, clinical outcomes, and determination of the optimal dose of peptide for further clinical trials. This study was approved by the Ethics Committee of the National Cancer Center and conformed to the ethical guidelines of the 1975 Declaration of Helsinki. The trial has been registered with the University Hospital Medical Information Network Clinical Trials Registry (UMIN-CTR number, 000001395).

Evaluation of toxicity and clinical response

Patients were evaluated for signs of toxicity during and after vaccination. Adverse events were graded according to the Common Terminology Criteria for Adverse Events v3.0 (CTCAE). Hematologic examinations were conducted before each vaccination. The tumor size was evaluated by computed tomography (CT) or MRI before vaccination, and then 1 month after the third vaccination. Tumor responses were evaluated according to the RECIST guidelines and the modified RECIST (mRECIST) assessment (22).

Measurement of immunologic response

Ex vivo IFN-γ enzyme-linked immunospot assay.

An ex vivo IFN-γ enzyme-linked immunospot (ELISPOT) assay was conducted to measure the antigen-specific CTL response, as described previously (21). Briefly, peripheral blood (30 mL) was obtained from each patient before the first vaccination and 2 weeks after each vaccination and centrifuged with a Ficoll-Paque gradient. PBMCs were frozen before immunologic analysis. All PBMCs obtained from an individual patient were incubated in the same plate and analyzed by ex vivo IFN-γ ELISPOT assay at the same time. Noncultured PBMCs (5 × 105 per well) were added to plates in the presence of peptide antigens (10 μg/mL) and incubated for 20 hours at 37°C in 5% CO2. The GPC3 antigen was the HLA-A2–restricted GPC3144–152 (FVGEFFTDV) peptide or HLA-A*24:02–restricted GPC3298–306 peptide (EYILSLEEL). PBMCs plus HLA-A2–restricted HIV19–27 (TLNAWVKVV) peptide (ProImmune) or HLA-A*24:02–restricted HIV583–591 (RYLKDQQLL; ProImmune) were used as negative controls. The assays were conducted in duplicate.

Dextramer staining and flow cytometric analysis.

The PBMCs were stained with HLA-A*02:01 Dextramer-RPE [GPC3144–152 (FVGEFFTDV), HIV19–27 (TLNAWVKVV); Immudex] and HLA-A*24:02 Dextramer-RPE [GPC3298–306 (EYILSLEEL), HIV583–591 (RYLKDQQLL); Immudex] for 10 minutes at room temperature and with anti-CD8-FITC (ProImmune) for 20 minutes at 4°C. Flow cytometry was carried out using a FACSAria cell sorter (BD Biosciences), as described previously (21).

Immunohistochemical analysis.

Biopsy specimens were taken from some of the vaccinated patients, each of whom provided informed consent. Specimens were stained with hematoxylin and eosin or monoclonal antibodies against GPC3 (clone 1G12; dilution 1:300; BioMosaics), CD8 (clone 1A5; dilution 1:80; Novocastra), HLA class I (clone EMR8/5; dilution 1:2,500; Hokudo), according to the manufacturers' directions.

GPC3 double-determinant (sandwich) ELISA.

Double-determinant (sandwich) ELISA of GPC3 was carried out as described previously (10). The serum-soluble protein GPC3 was detected by indirect ELISA using an anti-human GPC3 monoclonal antibody (clone 1G12; BioMosaics Inc.), and anti-human GPC3 sheep polyclonal antibody (R&D Systems), and recombinant human GPC3 (#211-GP/CF; R&D Systems).

Statistical analysis

OS rates were analyzed by the Kaplan–Meier method. Prognostic factors were evaluated using the log-rank test and Cox proportional hazard models. All statistical analyses were conducted using the PASW Statistics software, version 18.0 (SPSS Inc.). Statistical significance was defined by a value of P less than 0.05.

Patient characteristics

Thirty-three patients were enrolled in this study (Table 1). None of the patients dropped out because of adverse events caused by peptide vaccination. Two patients (cases 4 and 6) discontinued the regimen after the second vaccination because of liver function impairment resulting from tumor progression. One patient (case 28) could not undergo a CT scan after the third vaccination because of tumor progression. These patients were judged to have disease progression, but were not removed from the analyses at the advice of the effect and safety evaluation committee, including the external members. All patients received adequate follow-up to monitor toxicity. The median follow-up period was 9.0 months (range, 1.1–34.1 months). Of the 33 patients, 28 were male. Their average age was 64.3 years (range, 42–77 years). Five patients had a performance status (PS) of 1; all others had a PS of 0. Staging was conducted according to the tumor-node-metastasis (TNM) classification for HCC (Union for International Cancer Control). Sixteen patients were diagnosed with stage IV disease. Seven patients had Child–Pugh class B disease, and all others Child–Pugh class A disease. Twenty-three patients (70%) had a hepatic virus infection. All but 2 of the 33 patients had undergone conventional chemotherapy, surgery, and transcatheter arterial embolization before receiving GPC3 peptide vaccine therapy. At the time of the trial's initiation, sorafenib had not been approved by the drug administration in Japan. Only a few patients had received sorafenib as prior therapy in this phase I trial. One patient treated with gemcitabine had had stable disease for 5 months immediately before vaccination (case 33). The gemcitabine therapy was discontinued because of nausea and lightheadedness. Other patients had undergone prior therapy, but all of them showed progression of the disease before enrollment in this study.

Table 1.

Patient characteristics, clinical response, and GPC3-specific CTL response

The spot number of GPC3-specific CTLeExpression in the primary tumorf
Dose of peptide, mgNo.Age/sexStagea (UICC/LCSGJ)PSChild–PughHepatic virus infectionbPrior therapycTumor responsedPFS, moOs, moHLA-APrevaccinePostvaccineIncreased CTLGPC3HLA class I
0.3 75/M II III TAE, PEI, RFA, S-1 PD 2402 1+ 1+ 
 77/M IV IVB PEI, Proton, TAE, TAI SD 11 2402 1+ 1+ 
 67/M IV IVB — Ope SD 0206/0207 22 20 − 2+ 1+ 
 51/M IIIA IVA Ope, TAE, TAI PD 0201 NA NA 
 62/M IIIA III — TAI PD 0201 1+ 1+ 
 69/M IV IVB —  PD 0201 10 − NA NA 
 59/M IIIA IVA Ope, TAE, TAI PD 2402 1+ 1+ 
 55/M IIIA III MCT, PEI, TAE, RT, Sor, S-1 SD 17 0201 1+ 1+ 
1.0 68/F IIIC IVA PEI, TAE, RFA SD 13 0201 − NA NA 
 10 72/M IIIA IVA Ope, MCT, RFA, PEI, Sor, TAE, RT, S-1 SD 0201 51 1+ 1+ 
 11 60/M IIIC IVA TAE, RFA SD 2402 11 − 1+ 
 12 62/M II III — RFA, PEI, TAE PD 0201/0206 12 − 1+ 
 13 44/M IV IVB TAE, RFA, PEI, RT PD 24 2402 73 1+ 2+ 
 14 42/F IV IVB —  SD 14 2402 132 2+ 1+ 
3.0 15 67/F IV IVB — Ope, PEI, TAE, Proton SD 0201 23 1+ 1+ 
 16 58/M IIIA III — Ope, TAE, S-1, TAE SD 0201 101 1+ 1+ 
 17 75/M IIIC IVA RFA, TAE PD 2402 69 − 1+ 
 18 70/M IV IVB Ope, RT SD 14 2402 72 1+ 1+ 
 19 76/M IIIA III Ope, TAE, TAI SD 2402 31 68 1+ 1+ 
 20 73/M II II — Ope, TAE SD >34 2402 124 1+ 1+ 
10 21 52/M IV IVB Ope, TAE, S-1 SD 0201 100 2+ 1+ 
 22 71/M IIIC IVA — Ope SD >32 2402 171 − 1+ 
 23 70/M IV IVB Ope, TAI, TAE, PEI PD 0201 1+ − 
 27 56/M IV IVB TAE, UFT SD >23 2402 64 69 NA NA 
 28 57/M IIIA IVA TAE, RFA, TAI PD 2402 NA NA 
 29 68/M IIIA IVA Ope, TAE, TAI PD 0201 125 1+ 2+ 
 33 76/M IV IVB Ope, TAE, MCT, RFA, GEM SD >16 2402 2+ 1+ 
30 24 75/F IV IVB Ope, RFA, RT PR 12 0207 11 196 1+ 1+ 
 25 52/M IV IVB Ope, RFA, TAE, RT, UFT PD 12 0206 151 2+ 2+ 
 26 75/F II II MCT, RFA, TAE, TAI SD 2402 16 NA NA 
 30 69/M IV IVB — Ope, TAI, UFT, GEM+CDDP, RT SD 2402 34 1+ − 
 31 53/M IV IVB TAE, RFA SD 14 2402 NA NA 
 32 67/M IV IVB Ope, Sor, TAE PD >17 0201 441 − − 
The spot number of GPC3-specific CTLeExpression in the primary tumorf
Dose of peptide, mgNo.Age/sexStagea (UICC/LCSGJ)PSChild–PughHepatic virus infectionbPrior therapycTumor responsedPFS, moOs, moHLA-APrevaccinePostvaccineIncreased CTLGPC3HLA class I
0.3 75/M II III TAE, PEI, RFA, S-1 PD 2402 1+ 1+ 
 77/M IV IVB PEI, Proton, TAE, TAI SD 11 2402 1+ 1+ 
 67/M IV IVB — Ope SD 0206/0207 22 20 − 2+ 1+ 
 51/M IIIA IVA Ope, TAE, TAI PD 0201 NA NA 
 62/M IIIA III — TAI PD 0201 1+ 1+ 
 69/M IV IVB —  PD 0201 10 − NA NA 
 59/M IIIA IVA Ope, TAE, TAI PD 2402 1+ 1+ 
 55/M IIIA III MCT, PEI, TAE, RT, Sor, S-1 SD 17 0201 1+ 1+ 
1.0 68/F IIIC IVA PEI, TAE, RFA SD 13 0201 − NA NA 
 10 72/M IIIA IVA Ope, MCT, RFA, PEI, Sor, TAE, RT, S-1 SD 0201 51 1+ 1+ 
 11 60/M IIIC IVA TAE, RFA SD 2402 11 − 1+ 
 12 62/M II III — RFA, PEI, TAE PD 0201/0206 12 − 1+ 
 13 44/M IV IVB TAE, RFA, PEI, RT PD 24 2402 73 1+ 2+ 
 14 42/F IV IVB —  SD 14 2402 132 2+ 1+ 
3.0 15 67/F IV IVB — Ope, PEI, TAE, Proton SD 0201 23 1+ 1+ 
 16 58/M IIIA III — Ope, TAE, S-1, TAE SD 0201 101 1+ 1+ 
 17 75/M IIIC IVA RFA, TAE PD 2402 69 − 1+ 
 18 70/M IV IVB Ope, RT SD 14 2402 72 1+ 1+ 
 19 76/M IIIA III Ope, TAE, TAI SD 2402 31 68 1+ 1+ 
 20 73/M II II — Ope, TAE SD >34 2402 124 1+ 1+ 
10 21 52/M IV IVB Ope, TAE, S-1 SD 0201 100 2+ 1+ 
 22 71/M IIIC IVA — Ope SD >32 2402 171 − 1+ 
 23 70/M IV IVB Ope, TAI, TAE, PEI PD 0201 1+ − 
 27 56/M IV IVB TAE, UFT SD >23 2402 64 69 NA NA 
 28 57/M IIIA IVA TAE, RFA, TAI PD 2402 NA NA 
 29 68/M IIIA IVA Ope, TAE, TAI PD 0201 125 1+ 2+ 
 33 76/M IV IVB Ope, TAE, MCT, RFA, GEM SD >16 2402 2+ 1+ 
30 24 75/F IV IVB Ope, RFA, RT PR 12 0207 11 196 1+ 1+ 
 25 52/M IV IVB Ope, RFA, TAE, RT, UFT PD 12 0206 151 2+ 2+ 
 26 75/F II II MCT, RFA, TAE, TAI SD 2402 16 NA NA 
 30 69/M IV IVB — Ope, TAI, UFT, GEM+CDDP, RT SD 2402 34 1+ − 
 31 53/M IV IVB TAE, RFA SD 14 2402 NA NA 
 32 67/M IV IVB Ope, Sor, TAE PD >17 0201 441 − − 

Abbreviation: PD, progressive disease; PFS, progression-free survival; PS, performance status.

aStage: staging was carried out according to the TNM classification for HCC (Union for International Cancer Control, UICC) and the Japanese integrated staging system (Liver Cancer Study Group of Japan, LCSGJ).

bHepatic virus infection B. HBsAg was examined by radioimmunoassay. C: HCV was detected by RT-PCR.

cPrior therapy: Ope, surgery; TAE, transcatheter arterial embolization; PEI, percutaneous ethanol injection therapy; RFA, radiofrequency ablation; S-1, tegafur, gimeracil, oteracil potassium; proton, proton beam therapy; TAI, transcatheter arterial injection; RT, radiotherapy; Sor, sorafenib; MCT, microwave coagulation therapy; UFT, tegafur plus uracil; GEM, gemcitabine; CDDP, cis-diamminedichloroplatinum.

dTumor responses were evaluated according to RECIST guidelines and modified RECIST (mRECIST) assessment. The assessment of tumor response according to mRECIST was the same as that according to RECIST in all 33 patients.

eNumber of GPC3-specific CTL spots. The number of GPC3 peptide–specific CTL spots (postvaccination) was the maximum number of spots in an ex vivo IFN-γ ELISPOT assay for GPC3 peptide, carried out after vaccination and using 5 × 105 PBMCs.

fExpression of GPC3 and HLA class I was determined by immunohistochemistry. Degree of staining of tumor cells for GPC3: −, no reactivity; 1+, weak reactivity; 2+, strong reactivity; NA, not analyzed. Degree of staining of tumor cells for HLA class I: −, no membranous reactivity; 1+, weak membranous reactivity; 2+, strong membranous reactivity; NA, not analyzed.

We evaluated the expression of GPC3 and HLA class I in the primary tumors that could be obtained (Supplementary Fig. S1). GPC3 expression was detected in 21 of 26 patients (81%), consistent with previous reports (10–14). Cell membrane expression of HLA class I was evident in 23 of 26 patients (88%; Table 1).

GPC3 peptide vaccine was well-tolerated

The adverse events observed in this trial are listed in Table 2. Dose-limiting toxicity and dose-specific adverse events were not seen. Grade III hematologic adverse events (impaired liver function) were observed in 4 patients (cases 4, 6, 7, and 23). These 4 patients had progressively massive liver tumors. The effect and safety evaluation committee, including the external members, judged that these events were not related to the treatment, but rather to disease progression. All patients experienced grades I or II local skin reactions at the injection site. Transient immune-related events, including drug fever, rash, and flushing, were observed in most patients. Crotamiton, a scabicidal and antipruritic agent, was prescribed to the 5 patients who had mild itching, but no antipyretic analgesics were prescribed. These results suggest that GPC3 peptide vaccine therapy was well-tolerated.

Table 2.

The incidence of adverse event

Adverse eventTotal (%)Grade I (%)Grade II (%)Grade III (%)
Any event 33 (100) 9 (27.3) 20 (60.6) 4 (12.1) 
Any immune-related event 33 (100) 27 (81.8) 6 (18.2) 
 Drug fever 8 (24.2) 4 (12.1) 4 (12.1) 
 Rash or flushing 27 (81.8) 24 (72.7) 3 (9.1) 
 Injection site reaction 33 (100) 33 (100) 
 Pruritus 6 (18.2) 6 (18.2) 
Blood 15 (45.4) 6 (18.2) 9 (27.3) 
 Leukopenia 6 (18.2) 2 (6.1) 4 (12.1) 
 Neutropenia 8 (24.2) 5 (15.2) 3 (9.1) 
 Anemia 5 (15.2) 2 (6.1) 3 (9.1) 
 Thrombopenia 3 (9.1) 1 (3.0) 2 (6.1) 
 Increase in PT-INR 2 (6.1) 2 (6.1) 
Hepatic 23 (69.7) 10 (30.3) 9 (27.3) 4 (12.1) 
 Hyperbilirubinemia 9 (27.3) 3 (9.1) 4 (12.1) 2 (6.1) 
 Increase in aspartate aminotransferase 14 (42.4) 4 (12.1) 6 (18.2) 4 (12.1) 
 Increase in alanine aminotransferase 12 (36.4) 10 (30.3) 1 (3.0) 1 (3.0) 
Renal 9 (27.3) 6 (18.2) 3 (9.1) 
 Increase in creatinine 4 (12.1) 2 (6.1) 2 (6.1) 
 Proteinuria 6 (18.2) 4 (12.1) 2 (6.1) 
Other laboratory     
 Increase in alkaline phosphatase 9 (27.3) 4 (12.1) 4 (12.1) 1 (3.0) 
 Hypoalubuminemia 10 (30.3) 7 (21.2) 3 (9.1) 
 Hyponatremia 13 (39.4) 12 (36.4) 1 (3.0) 
 Hyperkalemia 4 (12.1) 4 (12.1) 
Adverse eventTotal (%)Grade I (%)Grade II (%)Grade III (%)
Any event 33 (100) 9 (27.3) 20 (60.6) 4 (12.1) 
Any immune-related event 33 (100) 27 (81.8) 6 (18.2) 
 Drug fever 8 (24.2) 4 (12.1) 4 (12.1) 
 Rash or flushing 27 (81.8) 24 (72.7) 3 (9.1) 
 Injection site reaction 33 (100) 33 (100) 
 Pruritus 6 (18.2) 6 (18.2) 
Blood 15 (45.4) 6 (18.2) 9 (27.3) 
 Leukopenia 6 (18.2) 2 (6.1) 4 (12.1) 
 Neutropenia 8 (24.2) 5 (15.2) 3 (9.1) 
 Anemia 5 (15.2) 2 (6.1) 3 (9.1) 
 Thrombopenia 3 (9.1) 1 (3.0) 2 (6.1) 
 Increase in PT-INR 2 (6.1) 2 (6.1) 
Hepatic 23 (69.7) 10 (30.3) 9 (27.3) 4 (12.1) 
 Hyperbilirubinemia 9 (27.3) 3 (9.1) 4 (12.1) 2 (6.1) 
 Increase in aspartate aminotransferase 14 (42.4) 4 (12.1) 6 (18.2) 4 (12.1) 
 Increase in alanine aminotransferase 12 (36.4) 10 (30.3) 1 (3.0) 1 (3.0) 
Renal 9 (27.3) 6 (18.2) 3 (9.1) 
 Increase in creatinine 4 (12.1) 2 (6.1) 2 (6.1) 
 Proteinuria 6 (18.2) 4 (12.1) 2 (6.1) 
Other laboratory     
 Increase in alkaline phosphatase 9 (27.3) 4 (12.1) 4 (12.1) 1 (3.0) 
 Hypoalubuminemia 10 (30.3) 7 (21.2) 3 (9.1) 
 Hyponatremia 13 (39.4) 12 (36.4) 1 (3.0) 
 Hyperkalemia 4 (12.1) 4 (12.1) 

Abbreviation: PT-INR, prothrombin time-international normalized ratio.

GPC3 peptide vaccination could induce peptide-specific CTLs in most patients

To determine whether the GPC3 peptide vaccine could induce a specific immune response, PBMCs, obtained from all patients before and after vaccination, were examined by ex vivo IFN-γ ELISPOT assay. After the second vaccination, the number of GPC3 peptide–specific CTLs in 5 × 105 PBMCs was increased from 0 to 441 in case 32 (Fig. 1A). As shown in Table 1, we found that the GPC3 peptide vaccine induced a GPC3-specific CTL response in 30 of the 33 patients (91%). GPC3-specific CTL frequency increased in a peptide dose-dependent manner (Fig. 1B). Generally, CTLs for some tumor antigens cannot be directly detected ex vivo; they can only be detected after expansion by repeated in vitro stimulation with the antigenic peptide on appropriate antigen-presenting cells. This finding can be attributed to the sensitivity of the assay and the low frequency of tumor antigen–specific CTLs (23). Surprisingly, GPC3-specific CTLs were directly detected ex vivo without in vitro peptide stimulation in almost all patients after GPC3 peptide vaccination.

Figure 1.

Immunologic monitoring of GPC3 peptide–specific T-cell responses. A, ex vivo IFN-γ ELISPOT assay for GPC3 in 5 × 105 PBMCs was carried out before and after vaccination in case 32. The Δspot number indicates the number of GPC3 peptide–specific CTLs. The number of IFN-γ–positive spots increased from 0 to 441 in the wells preincubated with GPC3 peptide. B, median spot number in ex vivo IFN-γ ELISPOT assay for GPC3 for each peptide dosage. GPC3-specific CTL frequency increased in a peptide dose-dependent manner. C, ex vivo GPC3 Dextramer staining before and after vaccination in case 32. GPC3 peptide–specific CTL frequency is indicated as the percentage of Dextramer-positive CTLs among PBMCs. The frequency of GPC3 peptide–specific CTLs increased from 0% to 0.12% in case 32. D, immunohistochemical staining showing CD8-positive lymphocytes infiltrating tumors before and after vaccination. In cases 8 and 11, CD8-positive T cells (brown) did not infiltrate the tumors before vaccination; in contrast, many CD8-positive T cells infiltrated the tumor after vaccination. Magnification, ×200.

Figure 1.

Immunologic monitoring of GPC3 peptide–specific T-cell responses. A, ex vivo IFN-γ ELISPOT assay for GPC3 in 5 × 105 PBMCs was carried out before and after vaccination in case 32. The Δspot number indicates the number of GPC3 peptide–specific CTLs. The number of IFN-γ–positive spots increased from 0 to 441 in the wells preincubated with GPC3 peptide. B, median spot number in ex vivo IFN-γ ELISPOT assay for GPC3 for each peptide dosage. GPC3-specific CTL frequency increased in a peptide dose-dependent manner. C, ex vivo GPC3 Dextramer staining before and after vaccination in case 32. GPC3 peptide–specific CTL frequency is indicated as the percentage of Dextramer-positive CTLs among PBMCs. The frequency of GPC3 peptide–specific CTLs increased from 0% to 0.12% in case 32. D, immunohistochemical staining showing CD8-positive lymphocytes infiltrating tumors before and after vaccination. In cases 8 and 11, CD8-positive T cells (brown) did not infiltrate the tumors before vaccination; in contrast, many CD8-positive T cells infiltrated the tumor after vaccination. Magnification, ×200.

Close modal

We also analyzed the GPC3-specific CTL frequency by flow cytometry using the GPC3 peptide, Dextramer. The GPC3-specific CTL frequency is indicated as the percentage of both Dextramer-positive and CD8-positice cells before and after vaccination, as shown in Fig. 1C. After the second vaccination, the frequency of GPC3-specific CTLs increased from 0% to 0.12% in case 32.

In many patients who were vaccinated only 3 times, the GPC3-specific CTL frequency decreased within 2 months after the third vaccination. We could vaccinate 4 or more times in 12 cases. In 9 of these, the GPC3-specific CTL frequency increased after the fourth vaccination (data not shown).

CTLs infiltrated the tumor after GPC3 peptide vaccination

Tumor biopsy was carried out (with informed consent) in 7 patients to evaluate the therapeutic effect after vaccination. We evaluated infiltration of CD8-positive T cells by immunohistochemical staining. In case 8, liver biopsy was carried out before and after vaccination. In case 11, neck lymph node metastasis was resected after vaccination. The specimen was compared with an abdominal lymph node metastasis sample obtained by a diagnostic biopsy that this patient underwent before vaccination. While CD8-positive T cells did not infiltrate the tumor before vaccination, marked infiltration of CD8-positive T cells into the tumor was observed after vaccination in both cases (Fig. 1D). In 5 of 7 cases, infiltration of CD8-positive T cells into the tumor was increased after vaccination.

Clinical responses

Patient characteristics and clinical responses in relation to GPC3-specific CTLs are shown in Table 1. Among the 33 patients, one (case 24) was judged to have a partial response (PR) and 19 patients stable disease (SD) for 2 months, according to RECIST. The assessment of tumor response according to mRECIST was the same as that according to RECIST in all 33 patients. The disease control rate (PR + SD) was 60.6% after 2 months. The median time to tumor progression (TTP) was 3.4 months [95% confidence interval (CI), 2.1–4.6]. The median OS was 9.0 months (95% CI, 8.0–10.0).

In case 24, supraclavicular lymph node metastases markedly regressed, 2 liver tumors disappeared, and the thoracic bone metastasis showed necrosis after the third vaccination (Fig. 2A and B). We carried out a biopsy of the remaining liver tumor and the thoracic bone metastasis after obtaining informed consent. Immunohistochemical staining showed expression of GPC3 and HLA class I on cells in the remaining liver tumor (Fig. 2C). Surprisingly, we detected massive infiltration of CD8-positive T cells into the remaining liver tumor by immunohistochemical staining. No viable tumor cells were found in the biopsy specimens of the thoracic bone metastasis.

Figure 2.

Response assessment in case 24. A, CT imaging, showing liver, pleura, and supraclavicular lymph node metastases before vaccination. B, CT imaging after vaccination was judged as an indicator of a PR. The supraclavicular lymph node metastasis and multiple liver tumors regressed markedly. The pleura metastasis was necrotic. C, we biopsied the remaining liver tumor after vaccination. Immunohistochemical staining showed expression of GPC3 and HLA class I on tumor cells. There was massive infiltration of CD8-positive T cells. Magnification, ×200.

Figure 2.

Response assessment in case 24. A, CT imaging, showing liver, pleura, and supraclavicular lymph node metastases before vaccination. B, CT imaging after vaccination was judged as an indicator of a PR. The supraclavicular lymph node metastasis and multiple liver tumors regressed markedly. The pleura metastasis was necrotic. C, we biopsied the remaining liver tumor after vaccination. Immunohistochemical staining showed expression of GPC3 and HLA class I on tumor cells. There was massive infiltration of CD8-positive T cells. Magnification, ×200.

Close modal

Four other patients (cases 1, 15, 16, and 17) had tumor necrosis or partial tumor reduction that did not meet the PR criteria.

Serum levels of α-fetoprotein (AFP) and des-γ-carboxy prothrombin (DCP) are useful tumor markers of HCC (24). The levels of AFP or DCP decreased temporarily at least once in 9 of the 33 patients during the 2-month period (Supplementary Table S1). In 7 of these 9 patients, the levels of DCP fell to less than 30% of baseline values. In 15 of 32 patients, GPC3 protein was detectable in serum before vaccination. The serum levels of GPC3 temporarily decreased at least once in 12 of these 15 patients (data not shown).

These results suggest that there is not the duration of the responses in regards to CTL induction and tumor responses in this phase I trial.

OS was correlated with GPC3-specific CTL frequency

We also examined prognostic factors (Table 3). Fifty GPC3 peptide–specific CTL spots were detected in an ex vivo IFN-γ ELISPOT assay conducted using 5 × 105 PBMCs, which means that the GPC3 peptide–specific CTL frequency in peripheral lymphocytes was is 1 × 10−4%. We focused on these 50 spots to elucidate prognostic factors. Univariate analysis indicated that distant metastasis (−; P = 0.032), invasion of the inferior vena cava (IVC) or portal vein (PV; P = 0.040), AFP ≥ 100 ng/mL (P = 0.003), tumor size ≥ 10 cm (P = 0.003), and GPC3-specific CTL frequency < 50 were prognostic factors for OS. Furthermore, AFP ≥ 100 ng/mL (P = 0.004; HR = 4.66; 95% CI, 1.61–13.19), tumor size ≥ 10 cm (P = 0.003; HR = 4.36; 95% CI, 1.58–12.05), and GPC3-specific CTL frequency < 50 (P = 0.032; HR = 2.71; 95% CI, 1.09–6.72) were prognostic factors for OS in a multivariate analysis. We showed that GPC3-specific CTL frequency could be a predictive marker of the effects of GPC3 peptide vaccination. We compared patients with GPC3-specfic CTL frequencies ≥ 50 (N = 15) with those with GPC3-specific CTL frequencies < 50 (N = 18) and found that there was no significant difference in clinical background. We only found a significant difference (P = 0.004) for vaccine consumption (≥1.0 vs. <1.0 mg; Supplementary Table S2). Analysis of all 33 patients showed that the median OS was 12.2 months (95% CI, 6.5–18.0) in patients with GPC3-specfic CTL frequencies ≥50, compared with 8.5 months (95% CI, 3.7–13.1) in those with GPC3-specfic CTL frequencies <50 (P = 0.033; Fig. 3).

Figure 3.

Kaplan–Meier curves for OS. Patients with GPC3-specfic CTL frequencies ≥50 had a longer survival than those with GPC3-specfic CTL frequencies <50 (P = 0.033). MST, median survival time.

Figure 3.

Kaplan–Meier curves for OS. Patients with GPC3-specfic CTL frequencies ≥50 had a longer survival than those with GPC3-specfic CTL frequencies <50 (P = 0.033). MST, median survival time.

Close modal
Table 3.

Prognostic factors of OS

P univariateP multivariateHR (95% CI)
Sex (male/female) 0.991   
Age (≥65/<65) 0.608   
Performance status (0/1) 0.707   
Child–Pugh (A/B) 0.063   
Virus infection (+/−) 0.956   
Distant metastasis (+/−) 0.032 0.284 1.71 (0.64–4.54) 
Invasion of IVC or PV (+/−) 0.040 0.706 1.21 (0.45–3.30) 
AFP (≥100/<100 ng/mL) 0.003 0.004 4.66 (1.61–13.19) 
Tumor sizea (≥10/<10 cm) 0.003 0.005 4.36 (1.58–12.05) 
GPC3-specific CTLb (≥50/<50) 0.033 0.032 2.71 (1.09–6.72) 
HLA (A2/A24) 0.091   
Vaccinec (≥1/<1 mg) 0.053   
P univariateP multivariateHR (95% CI)
Sex (male/female) 0.991   
Age (≥65/<65) 0.608   
Performance status (0/1) 0.707   
Child–Pugh (A/B) 0.063   
Virus infection (+/−) 0.956   
Distant metastasis (+/−) 0.032 0.284 1.71 (0.64–4.54) 
Invasion of IVC or PV (+/−) 0.040 0.706 1.21 (0.45–3.30) 
AFP (≥100/<100 ng/mL) 0.003 0.004 4.66 (1.61–13.19) 
Tumor sizea (≥10/<10 cm) 0.003 0.005 4.36 (1.58–12.05) 
GPC3-specific CTLb (≥50/<50) 0.033 0.032 2.71 (1.09–6.72) 
HLA (A2/A24) 0.091   
Vaccinec (≥1/<1 mg) 0.053   

aTumor size estimated by the RECIST.

bThe GPC3 peptide–specific CTL frequency examined with ex vivo IFN-γ ELISPOT assay in 5 × 105 PBMCs.

cThe dosage of one vaccine.

We did not observe dose-limiting toxicity in this study. It was difficult to determine the maximum tolerated dose of peptide. A peptide dose of greater than 1.0 mg was required for adequate induction of GPC3-specific CTLs. However, it was complicated to inject more than 10 mg of peptide intradermally because injection mixtures contained both peptide and IFA, and doses of peptide vaccine >10 mg emulsified with IFA (consisting of 2 mL of fluid, including 1 mL of IFA), increased local skin reactions (induration, blushing) at the injection site (Supplementary Fig. S2). Therefore, a dose of peptide of 3.0 mg is recommended for future clinical trials.

We evaluated the expression of GPC3 in the primary tumors of 26 patients by immunohistochemistry. Among the 21 patients with low GPC3 expression (degree of staining − or 1+), one patient was judged to have a PR, and 3 patients have shown long-term survival. We do not suggest that only patients with high GPC3 expression (degree of staining 2+) should be enrolled in further clinical trials.

We studied immunologic responses using an ex vivo IFN-γ ELISPOT assay. The GPC3 peptide vaccine induced GPC3-specific CTL responses in 30 of the 33 patients. In contrast, clear immune responses were not observed in patients with HCC in another vaccination trial (9). Differences in tumor antigen may account for the differences in immune response between the 2 vaccination trials. Previous studies have shown that GPC3 is also overexpressed in other malignant tumors, including melanomas, Wilms' tumor, hepatoblastoma, ovarian clear cell carcinoma, and lung squamous cell carcinoma (12, 25–28). GPC3 might also be an effective target for immunotherapy against these tumors (29, 30).

In our study, none of the patients in the 0.1 mg dose group showed more than 50 GPC3 peptide–specific CTL spots. GPC3-specific CTL frequency increased in a peptide dose-dependent manner. Previously, Salgaller and colleagues reported no dose dependency in the capacity of the gp100 peptide to enhance immunogenicity in humans (at doses 1.0–10 mg; ref. 31). In contrast, our data indicate dose dependency in CTL induction, consistent with a previous report using a mouse model (20).

Ten of the 25 patients who received a dose higher than 1.0 mg did not exhibit GPC3-specfic CTL frequencies ≥50. There was no significant difference in the clinical background of patients with GPC3-specific CTL frequencies ≥50 and those with <50. However, GPC3-specific CTL frequency tended to correlate with the serum level of AFP or summed intrahepatic tumor size (Supplementary Table S2). In this study, several patients with advanced HCC exhibited a poor immunologic response to GPC3 peptide vaccination. There are several possible explanations for this poor immunogenicity. HCC is frequently accompanied by cirrhosis, which creates an immunosuppressive environment. There is impairment of the function and maturation of dendritic cells, which has been shown to be related to an imbalance in the extracellular amino acid profile (32). In progressive HCC, the induction of CTL may be suppressed by regulatory T cells or immunosuppressive cytokines (33). It has been reported that GPC3-specific CTLs become exhausted in HCC, and that this exhausted state cannot be reversed by blocking the CTLA-4 and PD-1 inhibitory costimulation pathways (34). Further studies will be necessary to increase the clinical efficacy of immunotherapy for advanced HCC.

The primary endpoint of this study was assessment of the safety of vaccination, but we also showed that tumor antigen–specific CTLs had a crucial role in the immunotherapy against GPC3. GPC3-specific CTL frequency was correlated with OS in this study. Peptide-specific IgG and delayed-type hypersensitivity postvaccination have been reported as potential predictive makers of prolonged survival in patients with advanced cancer vaccinated with peptides (35, 36). However, correlations between immune responses and OS have not been reported in other immunotherapy trials for HCC (7–9, 37). We found that patients with GPC3-specfic CTL frequencies ≥ 50 had a longer survival than those with GPC3-specfic CTL frequencies <50. There was no significant difference in the clinical backgrounds of patients with GPC3-specific CTL frequencies ≥50 and those with <50.

We clearly showed the presence of GPC3 peptide–specific CTLs in peripheral blood, and showed that many CD8-positive T cells infiltrated tumors after GPC3 peptide vaccination. The evidence in this study serves as a proof-of-concept for immunotherapy using tumor antigen–specific CTLs. However, we did not confirm that the tumor-infiltrating lymphocytes detected after vaccination were GPC3 peptide–specific CTLs. We are currently initiating a pilot study of liver biopsies carried out before and after GPC3 peptide vaccination for advanced HCC to determine whether tumor-infiltrating lymphocytes are indeed GPC3 peptide–specific CTLs.

No complete responses were observed when GPC3 peptide vaccination was used as the sole therapy for advanced HCC. To-date, there has been no report of an adequate antitumor efficacy of immunotherapy in clinical trials involving patients with advanced HCC; however, immunotherapy, as an adjuvant after surgical resection, is expected (38). On the basis of this study, we have begun a phase II study of the GPC3-derived peptide vaccine as an adjuvant therapy for patients with HCC and have also planned combinatorial approaches with chemotherapy.

In conclusion, this phase I clinical trial of a GPC3-derived peptide vaccine showed the vaccination to be safe and indicated a plethora of immunologic responses. This study also showed that GPC3-specific CTL frequency was correlated with OS in patients with advanced HCC who received the GPC3 peptide vaccine.

No potential conflicts of interest were disclosed.

Conception and design: T. Kuronuma, T. Takayama, K. Uesaka, J. Furuse, T. Nakatsura

Development of methodology: J. Furuse

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Y. Sawada, T. Yoshikawa, D. Nobuoka, H. Shirakawa, Y. Motomura, H. Ishii, K. Nakachi, M. Konishi, S. Takahashi, N. Gotohda, J. Furuse, T. Kinoshita, T. Nakatsura

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Y. Sawada, S. Mizuno, J. Furuse, T. Nakatsura

Writing, review, and/or revision of the manuscript: Y. Sawada, T. Yoshikawa, D. Nobuoka, T. Takayama, J. Furuse, T. Nakatsura

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J. Furuse

Study supervision: T. Nakagohri, T. Takayama, K. Yamao, J. Furuse

Monitoring and evaluation of data and safety of the study: K. Uesaka

The authors thank Manami Shimomura for technical assistance.

This study was supported in part by Health and Labor Science Research Grants for Research on Hepatitis and for Clinical Research from the Ministry of Health, Labor, and Welfare, Japan.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Parkin
DM
,
Bray
F
,
Ferlay
J
,
Pisani
P
. 
Global cancer statistics, 2002
.
CA Cancer J Clin
2005
;
55
:
74
108
.
2.
Llovet
JM
,
Ricci
S
,
Mazzaferro
V
,
Hilgard
P
,
Gane
E
,
Blanc
JF
, et al
Sorafenib in advanced hepatocellular carcinoma
.
N Engl J Med
2008
;
359
:
378
90
.
3.
Cheng
AL
,
Kang
YK
,
Chen
Z
,
Tsao
CJ
,
Qin
S
,
Kim
JS
, et al
Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma: a phase III randomised, double-blind, placebo-controlled trial
.
Lancet Oncol
2009
;
10
:
25
34
.
4.
Morimoto
M
,
Numata
K
,
Kondo
M
,
Hidaka
H
,
Takeda
J
,
Shibuya
A
, et al
Higher discontinuation and lower survival rates are likely in elderly Japanese patients with advanced hepatocellular carcinoma receiving sorafenib
.
Hepatol Res
2011
;
41
:
296
302
.
5.
Greten
TF
,
Manns
MP
,
Korangy
F
. 
Immunotherapy of hepatocellular carcinoma
.
J Hepatol
2006
;
45
:
868
78
.
6.
Mizukoshi
E
,
Nakamoto
Y
,
Arai
K
,
Yamashita
T
,
Sakai
A
,
Sakai
Y
, et al
Comparative analysis of various tumor-associated antigen-specific t-cell responses in patients with hepatocellular carcinoma
.
Hepatology
2011
;
53
:
1206
16
.
7.
Butterfield
LH
,
Ribas
A
,
Meng
WS
,
Vollmer
CM
,
Ribas
A
,
Dissette
VB
, et al
T-cell responses to HLA-A*0201 immunodominant peptides derived from alpha-fetoprotein in patients with hepatocellular cancer
.
Clin Cancer Res
2003
;
9
:
5902
8
.
8.
Butterfield
LH
,
Ribas
A
,
Dissette
VB
,
Lee
Y
,
Yang
JQ
,
De la Rocha
P
, et al
A phase I/II trial testing immunization of hepatocellular carcinoma patients with dendritic cells pulsed with four alpha-fetoprotein peptides
.
Clin Cancer Res
2006
;
12
:
2817
25
.
9.
Greten
TF
,
Forner
A
,
Korangy
F
,
N'Kontchou
G
,
Barget
N
,
Ayuso
C
, et al
A phase II open trial evaluating2288;safety and efficacy of a telomerase peptide vaccination in patients with advanced hepatocellular carcinoma
.
BMC Cancer
2010
;
10
:
209
.
10.
Nakatsura
T
,
Yoshitake
Y
,
Senju
S
,
Monji
M
,
Komori
H
,
Motomura
Y
, et al
Glypican-3, overexpressed specifically in human hepatocellular carcinoma, is a novel tumor marker
.
Biochem Biophys Res Commun
2003
;
306
:
16
25
.
11.
Capurro
M
,
Wanless
IR
,
Sherman
M
,
Deboer
G
,
Shi
W
,
Miyoshi
E
, et al
Glypican-3: a novel serum and histochemical marker for hepatocellular carcinoma
.
Gastroenterology
2003
;
125
:
89
97
.
12.
Nakatsura
T
,
Nishimura
Y
. 
Usefulness of the novel oncofetal antigen glypican-3 for diagnosis of hepatocellular carcinoma and melanoma
.
BioDrugs
2005
;
19
:
71
7
.
13.
Shirakawa
H
,
Kuronuma
T
,
Nishimura
Y
,
Hasebe
T
,
Nakano
M
,
Gotohda
N
, et al
Glypican-3 is a useful diagnostic marker for a component of hepatocellular carcinoma in human liver cancer
.
Int J Oncol
2009
;
34
:
649
56
.
14.
Shirakawa
H
,
Suzuki
H
,
Shimomura
M
,
Kojima
M
,
Gotohda
N
,
Takahashi
S
, et al
Glypican-3 expression is correlated with poor prognosis in hepatocellular carcinoma
.
Cancer Sci
2009
;
100
:
1403
7
.
15.
Nakatsura
T
,
Komori
H
,
Kubo
T
,
Yoshitake
Y
,
Senju
S
,
Katagiri
T
, et al
Mouse homologue of a novel human oncofetal antigen, glypican-3, evokes T-cell-mediated tumor rejection without autoimmune reactions in mice
.
Clin Cancer Res
2004
;
10
:
8630
40
.
16.
Komori
H
,
Nakatsura
T
,
Senju
S
,
Yoshitake
Y
,
Motomura
Y
,
Ikuta
Y
, et al
Identification of HLA-A2- or HLA-A24-restricted CTL epitopes possibly useful for glypican-3-specific immunotherapy of hepatocellular carcinoma
.
Clin Cancer Res
2006
;
12
:
2689
97
.
17.
Date
Y
,
Kimura
A
,
Kato
H
,
Sasazuki
T
. 
DNA typing of the HLA-A gene: population study and identification of four new alleles in Japanese
.
Tissue Antigens
1996
;
47
:
93
101
.
18.
Ohmori
M
,
Yasunaga
S
,
Maehara
Y
,
Sugimachi
K
,
Sasazuki
T
. 
DNA typing of HLA class I (HLA-A) and class II genes (HLA-DR, -DQ and -DP) in Japanese patients with gastric cancer
.
Tissue Antigens
1997
;
50
:
277
82
.
19.
Browning
M
,
Krausa
P
. 
Genetic diversity of HLA-A2: evolutionary and functional significance
.
Immunol Today
1996
;
17
:
165
70
.
20.
Motomura
Y
,
Ikuta
Y
,
Kuronuma
T
,
Komori
H
,
Ito
M
,
Tsuchihara
M
, et al
HLA-A2 and -A24-restricted glypican-3-derived peptide vaccine induce specific CTLs: preclinical study using mice
.
Int J Oncol
2008
;
32
:
985
90
.
21.
Yoshikawa
T
,
Nakatsugawa
M
,
Suzuki
S
,
Shirakawa
H
,
Nobuoka
D
,
Sakemura
N
, et al
HLA-A2-restricted glypican-3 peptide-specific CTL clones induced by peptide vaccine show high avidity and antigen-specific killing activity against tumor cells
.
Cancer Sci
2011
;
102
:
918
25
.
22.
Lencioni
R
,
Llovet
JM
. 
Modified RECIST(mRECIST) assessment for hepatocellular carcinoma
.
Sem Lis Dis
2010
;
30
:
52
60
.
23.
Romero
P
,
Cerottini
JC
,
Speiser
DE
. 
Monitoring tumor antigen specific T-cell responses in cancer patients and phase I clinical trials of peptide-based vaccination
.
Cancer Immunol Immunother
2004
;
53
:
249
55
.
24.
Nobuoka
D
,
Kato
Y
,
Gotohda
N
,
Takahashi
S
,
Nakagohri
T
,
Konishi
M
, et al
Postoperative serum α-fetoprotein level is a useful predictor of recurrence after hepatectomy for hepatocellular carcinoma
.
Oncol Rep
2010
;
24
:
521
8
.
25.
Saikali
Z
,
Sinnett
D
. 
Expression of glypican 3 (GPC3) in embryonal tumors
.
Int J Cancer
2000
;
89
:
418
22
.
26.
Toretsky
JA
,
Zitomersky
NL
,
Eskenazi
AE
,
Voigt
RW
,
Strauch
ED
,
Sun
CC
, et al
Glypican-3 expression in Wilms tumor and hepatoblastoma
.
J Pediatr Hematol Oncol
2001
;
23
:
496
9
.
27.
Maeda
D
,
Ota
S
,
Takazawa
Y
,
Aburatani
H
,
Nakagawa
S
,
Yano
T
, et al
Glypican-3 expression in clear cell adenocarcinoma of the ovary
.
Mod Pathol
2009
;
22
:
824
32
.
28.
Aviel-Ronen
S
,
Lau
SK
,
Pintilie
M
,
Lau
D
,
Liu
N
,
Tsao
MS
, et al
Glypican-3 is overexpressed in lung squamous cell carcinoma, but not in adenocarcinoma
.
Mod Pathol
2008
;
21
:
817
25
.
29.
Motomura
Y
,
Senju
S
,
Nakatsura
T
,
Matsuyoshi
H
,
Hirata
S
,
Monji
M
, et al
Embryonic stem cell–derived dendritic cells expressing glypican-3, recently identified oncofetal antigen, induce protective immunity against highly metastatic mouse melanoma, B16-F10
.
Cancer Res
2006
;
66
:
2414
22
.
30.
Suzuki
S
,
Yoshikawa
T
,
Hirosawa
T
,
Shibata
K
,
Kikkawa
F
,
Akatsuka
Y
, et al
Glypican-3 could be an effective target for immunotherapy combined with chemotherapy against ovarian clear cell carcinoma
.
Cancer Sci
2011
;
102
:
1622
9
.
31.
Salgaller
ML
,
Marincola
FM
,
Cormier
JN
,
Rosenberg
SA
. 
Immunization against epitopes in the human melanoma antigen gp100 following patient immunization with synthetic peptides
.
Cancer Res
1996
;
56
:
4749
57
.
32.
Kakazu
E
,
Ueno
Y
,
Kondo
Y
,
Fukushima
K
,
Shiina
M
,
Inoue
J
, et al
Branched chain amino acids enhance the maturation and function of myeloid dendritic cells ex vivo in patients with advanced cirrhosis
.
Hepatology
2009
;
50
:
1936
45
.
33.
Fu
J
,
Xu
D
,
Liu
Z
,
Shi
M
,
Zhao
P
,
Fu
B
, et al
Increased regulatory T cells correlate with CD8 T-cell impairment and poor survival in hepatocellular carcinoma patients
.
Gastroenterology
2007
;
132
:
2328
39
.
34.
Xub
Y
,
Lib
H
,
Gaob
RL
,
Adeyemo
O
,
Itkin
M
,
Kaplan
DE
. 
Expansion of interferon-gamma-producing multifunctional CD4+ T-cells and dysfunctional CD8+ T-cells by glypican-3 peptide library in hepatocellular carcinoma patients
.
Clin Immunol
2011
;
139
:
302
13
.
35.
Mine
T
,
Sato
Y
,
Noguchi
M
,
Sasatomi
T
,
Gouhara
R
,
Tsuda
N
. 
Humoral responses to peptide correlate with overall survival in advanced cancer patients vaccinated with peptides based on pre-existing, peptide-specific cellular responses
.
Clin Cancer Res
2004
;
10
:
929
37
.
36.
DiFronzo
LA
,
Gupta
RK
,
Essner
R
,
Foshag
LJ
,
O'Day
SJ
,
Wanek
LA
, et al
Enhanced humoral immune response correlates with improved disease-free and overall survival in American Joint Committee on Cancer stage II melanoma patients receiving adjuvant polyvalent vaccine
.
J Clin Oncol
2002
;
20
:
3242
8
.
37.
Palmer
DH
,
Midgley
RS
,
Mirza
N
,
Torr
EE
,
Ahmed
F
,
Steele
JC
, et al
A phase II study of adoptive immunotherapy using dendritic cells pulsed with tumor lysate in patients with hepatocellular carcinoma
.
Hepatology
2009
;
49
:
124
32
.
38.
Takayama
T
,
Sekine
T
,
Makuuchi
M
,
Yamasaki
S
,
Kosuge
T
,
Yamamoto
J
, et al
Adoptive immunotherapy to lower postsurgical recurrence rates of hepatocellular carcinoma: a randomised trial
.
Lancet
2000
;
356
:
802
7
.