Development of antigen-prediction algorithm for personalized neoantigen vaccine using human leukocyte antigen transgenic mouse.

Immunotherapy is currently recognized as the fourth modality in cancer therapy. CTL can detect cancer cells via complexes involving human leukocyte antigen (HLA) class I molecules and peptides derived from tumor antigens, resulting in antigen-specific cancer rejection. The peptides may be predicted in silico using machine learning-based algorithms. Neopeptides, derived from neoantigens encoded by somatic mutations in cancer cells, are putative immunotherapy targets, as they have high tumor specificity and immunogenicity. Here, we used our pipeline to select 278 neoepitopes with high predictive "SCORE" from the tumor tissues of 46 patients with hepatocellular carcinoma or metastasis of colorectal carcinoma. We validated peptide immunogenicity and specificity by in vivo vaccination with HLA-A2, A24, B35, and B07 transgenic mice using ELISpot assay, in vitro and in vivo killing assays. We statistically evaluated the power of our prediction algorithm and demonstrated the capacity of our pipeline to predict neopeptides (area under the curve = 0.687, P < 0.0001). We also analyzed the potential of long peptides containing the predicted neoepitopes to induce CTLs. Our study indicated that the short peptides predicted using our algorithm may be intrinsically present in tumor cells as cleavage products of long peptides. Thus, we empirically demonstrated that the accuracy and specificity of our prediction tools may be potentially improved in vivo using the HLA transgenic mouse model. Our data will help to design feedback algorithms to improve in silico prediction, potentially allowing researchers to predict peptides for personalized immunotherapy.

Peptide vaccine as an adjuvant therapy for glypican-3-positive hepatocellular carcinoma induces peptide-specific CTLs and improves long prognosis.

There is no established postoperative adjuvant therapy for hepatocellular carcinoma (HCC), and improvement of patient prognosis has been limited. We conducted long-term monitoring of patients within a phase II trial that targeted a cancer antigen, glypican-3 (GPC3), specifically expressed in HCC. We sought to determine if the GPC3 peptide vaccine was an effective adjuvant therapy by monitoring disease-free survival and overall survival. We also tracked GPC3 immunohistochemical (IHC) staining, CTL induction, and postoperative plasma GPC3 for a patient group that was administered the vaccine (n = 35) and an unvaccinated patient group that underwent surgery only (n = 33). The 1-y recurrence rate after surgery was reduced by approximately 15%, and the 5-y and 8-y survival rates were improved by approximately 10% and 30%, respectively, in the vaccinated group compared with the unvaccinated group. Patients who were positive for GPC3 IHC staining were more likely to have induced CTLs, and 60% survived beyond 5 y. Vaccine efficacy had a positive relationship with plasma concentration of GPC3; high concentrations increased the 5-y survival rate to 75%. We thus expect GPC3 vaccination in patients with HCC, who are positive for GPC3 IHC staining and/or plasma GPC3 to induce CTL and have significantly improved long-term prognosis.

Plasma and tumoral glypican-3 levels are correlated in patients with hepatitis C virus-related hepatocellular carcinoma.

Glypican-3 (GPC3) is a cancer antigen expressed in approximately 80% of hepatocellular carcinomas (HCC) and is secreted into the blood. To confirm the effectiveness of GPC3 as a biomarker in HCC, we analyzed the relationship between GPC3 expression levels in cancer cells and in blood in 56 patients with HCC. Preoperative plasma GPC3 levels were determined with an immunoassay, and expression of GPC3 in resected tumors was analyzed by immunohistochemical staining. Median plasma GPC3 level in all HCC cases was 4.6 pg/mL, and tended to be higher in patients with hepatitis C virus (HCV)-related HCC (HCV group) (9.9 pg/mL) than in patients with hepatitis B virus (HBV)-related HCC (HBV group) (2.6 pg/mL) or in those without virus infection (None group) (3.0 pg/mL), suggesting that the virus type most likely influences GPC3 secretion. Median percentage of GPC3+ cells in tumors was also higher in the HCV (26.2%) and HBV (11.1%) groups than in the None group (4.2%). In the HCV group, there was a positive correlation between the two parameters (r = 0.66, P < .01). Moreover, receiver operating characteristic analysis predicted >10% GPC3+ cells in a tumor if the cut-off value was 6.8 pg/mL (sensitivity 80%, specificity 100%; area under the curve 0.875, 95% confidence interval 0.726-1) in the HCV group. Plasma concentration of GPC3 could be a predictive marker of tumoral GPC3 expression in patients with HCV-related HCC, suggesting a useful biomarker for immunotherapies targeting GPC3, although larger-scale validations are needed.

Vaccination with liposome-coupled glypican-3-derived epitope peptide stimulates cytotoxic T lymphocytes and inhibits GPC3-expressing tumor growth in mice.

Because therapeutic manipulation of immunity can induce tumor regression, anti-cancer immunotherapy is considered a promising treatment modality. We previously reported that glypican-3 (GPC3), an oncofetal antigen overexpressed in hepatocellular carcinoma (HCC), is a useful target for cytotoxic T lymphocyte (CTL)-mediated cancer immunotherapy, and we have performed clinical trials using the GPC3-derived peptide vaccine. Although vaccine-induced GPC3-peptide-specific CTLs were often tumor reactive in vitro and were correlated with overall survival, no complete response was observed. In the current study, we synthesized liposome-coupled GPC3-derived CTL epitope peptide (pGPC3-lipsome) and investigated its antitumor potential. Vaccination with pGPC3-liposome induced peptide-specific CTLs at a lower dose than conventional vaccine emulsified in incomplete Freund's adjuvant. Coupling of pGPC3 to liposomes was essential for effective priming of GPC3-specific CTLs. In addition, immunization with pGPC3-liposome inhibited GPC3-expressing tumor growth. Thus, vaccination with tumor-associated antigen-derived epitope peptides coupled to the surfaces of liposomes may be a novel therapeutic strategy for cancer.

Peptide-Based Vaccines for Hepatocellular Carcinoma: A Review of Recent Advances.

Primary liver cancer is the sixth most commonly diagnosed cancer and the third leading cause of cancer-related deaths worldwide. After surgery, up to 70% of patients experience relapses. The current first-line therapy for advanced cases of hepatocellular carcinoma (HCC) comprises sorafenib and lenvatinib administered as single-drug therapies. Regorafenib, cabozantinib, and ramucirumab are administered as second-line therapies. Recently, it has been reported that using the immune checkpoint inhibitors atezolizumab (anti-PDL1 antibody) and bevacizumab (anti-VEGF antibody) leads to longer overall survival of unresectable cases, when compared with the use of sorafenib. The role of cancer immunity against HCC has attracted the attention of clinicians. In this review, we describe our phase I/II clinical trials of peptide vaccines targeting GPC3 in HCC and discuss the potential of peptide vaccines targeting common cancer antigens that are highly expressed in HCC, such as WT-I, AFP, ROBO1, and FOXM1. Further, we introduce recent cancer vaccines targeting neoantigens, which have attracted attention in recent times, as well as present our preclinical studies, the results of which might aid to initiate a neoantigen vaccine clinical trial, which would be the first of its kind in Japan.

Usefulness of plasma full-length glypican-3 as a predictive marker of hepatocellular carcinoma recurrence after radial surgery.

Predicting the risk of hepatocellular carcinoma (HCC) recurrence before treatment is necessary for developing subsequent treatment policies. Several tumor markers found in blood, such as alpha-fetoprotein (AFP) and protein induced by vitamin K absence or antagonist-II (PIVKA-II), are presently used to determine the occurrence and recurrence of HCC and to predict patient prognosis. However, these markers are insufficient for these purposes as certain patients have HCC recurrence despite exhibiting negative AFP and PIVKA-II. The present study identified glypican-3 (GPC3), an embryonal carcinoma antigen that is expressed specifically in HCC and is secreted into blood. Although the N-terminal domain of GPC3 in sera may be a potential prognostic factor for HCC, its biological role remains unclear. By contrast, full-length GPC3 (FL-GPC3) is reported to serve important roles in cell differentiation, proliferation and signaling events that cause HCC. Given the biological roles of FL-GPC3 in HCC progression, the present study evaluated its potential as a predictive marker of HCC recurrence. In the present study, a novel measurement system was constructed to specifically measure plasma FL-GPC3. Subsequently, its ability to predict recurrence after radical surgery in 39 HCC patients was evaluated. The results revealed that preoperative FL-GPC3 levels in patients with recurrence were significantly higher than those in patients without recurrence, suggesting that FL-GPC3 could be a better predictive maker of risk of recurrence than AFP or PIVKA-II. Furthermore, it was determined that the combination of FL-GPC3, AFP and PIVKA-II could predict recurrence within one year of radical surgery with high sensitivity and specificity. Based on these results, the validation of FL-GPC3 as a predictive marker of HCC recurrence in a larger population is warranted.

Usefulness of serum microRNA as a predictive marker of recurrence and prognosis in biliary tract cancer after radical surgery.

Biliary tract cancer (BTC) is an aggressive type of malignant tumour. Even after radical resection, the risk of recurrence is still high, resulting in a poor prognosis. Here, we investigated the usefulness of serum miRNAs as predictive markers of recurrence and prognosis for patients with BTC after radical surgery using 66 serum samples that were collected at three time points from 22 patients with BTC who underwent radical surgery. Using microarray analysis, we successfully identified six specific miRNAs (miR-1225-3p, miR-1234-3p, miR1260b, miR-1470, miR-6834-3p, and miR-6875-5p) associated with recurrence and prognosis of BTC after radical surgery. In addition, using a combination of these miRNAs, we developed a recurrence predictive index to predict recurrence in patients with BTC after operation with high accuracy. Patients having higher index scores (≥ cut-off) had significantly worse recurrence-free survival (RFS) and overall survival (OS) than those with lower index scores (

Immunological efficacy of glypican-3 peptide vaccine in patients with advanced hepatocellular carcinoma.

We have previously conducted a phase I trial to test the efficacy of a glypican-3 (GPC3) peptide vaccine in patients with advanced hepatocellular carcinoma (HCC); however, its immunological mechanism of action remains unclear. Here, we report a pilot study conducted to evaluate the immunological mechanisms of action of this GPC3 peptide vaccine (UMIN-CTR number 000005093). Eleven patients with advanced HCC were vaccinated with the GPC3 peptide in this trial. The primary end point was GPC3 peptide-specific immune response induced by the GPC3 peptide vaccination. The secondary endpoints were clinical and biologic outcomes. We demonstrated that the present vaccine induced GPC3 peptide-specific cytotoxic T lymphocytes (CTLs), which were found to infiltrate into the tumor. Moreover, we established GPC3 peptide-specific CTL clones from a biopsy specimen: these cells exhibited GPC3 peptide-specific cytokine secretion and cell cytotoxicity. The plasma GPC3 level tended to decrease temporarily at least once during the follow-up period. The GPC3-specific CTL frequency after vaccination was correlated with overall survival. The degree of skin reactions at the injection site correlated with the GPC3 peptide-specific CTLs. Furthermore, we sequenced the T cell receptors (TCRs) of tumor-infiltrating lymphocyte (TIL) clones, and confirmed the existence of this TCR repertoire in both tumor tissue and PBMCs. In response to these data, we are developing TCR-engineered T cell therapy using TCR sequences obtained from GPC3 peptide-specific CTL clones for improved efficacy in patients with advanced HCC.

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

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.

Hepatocellular carcinoma cell sensitivity to Vγ9Vδ2 T lymphocyte-mediated killing is increased by zoledronate.

The limited efficacy of vaccines in hepatocellular carcinoma (HCC), due to the low frequency of tumor-infiltrating cytotoxic T lymphocytes (CTLs), indicates the importance of innate immune surveillance, which assists acquired immunity by directly recognizing and eliminating HCC. Innate Vγ9Vδ2 T cells have major histocompatibility complex-unrestricted antitumor activity and are activated by phosphoantigens, which are upregulated in cancer cells by the nitrogen-containing bisphosphonate, zoledronate (Zol). A better understanding of HCC susceptibility to Zol and downstream γδ T cell-mediated killing is essential to optimize γδ T cell-mediated immunotherapy. This study systematically examined the interactions between γδ T cells and Zol-treated HCC cell lines (HepG2, HLE, HLF, HuH-1, JHH5, JHH7, and Li-7) in vitro. All HCC cell lines expressed the DNAX accessory molecule-1 ligands, poliovirus receptor, and Nectin-2, and γδ T cell-mediated killing of these cells was significantly enhanced by Zol. Small interfering RNA-mediated knockdown of these ligands did not affect the susceptibility to γδ T cell lysis. This killing activity was partly inhibited by mevastatin, an inhibitor of the mevalonate pathway, and markedly reduced by a monoclonal antibody to γ- and δ-chain T cell receptor, indicating that this is crucial for Zol-induced HCC killing. In addition, Zol-treated HCC cell lines triggered γδ T cell proliferation and induced production of Th1 and Th2, but not Th17, cytokines. The Zol concentration that enhanced HCC cell susceptibility to γδ T cell killing was lower than that required to directly inhibit HCC proliferation. Thus, γδ T cells may be important effector cells in the presence of Zol, especially where there are insufficient number of cancer antigen-specific CTLs to eliminate HCC. Our in vitro data support the proposal that Zol-treatment, combined with adaptive γδ T cell immunotherapy, may provide a feasible and effective approach for treatment of HCC.

Enhancement of antitumor effect by peptide vaccine therapy in combination with anti-CD4 antibody: Study in a murine model.

PURPOSE: The clinical efficacy of cancer peptide vaccine therapy is insufficient. To enhance the anti-tumor effect of peptide vaccine therapy, we combined this therapy with an anti-CD4 mAb (GK1.5), which is known to deplete CD4+ cells, including regulatory T cells (Tregs). METHODS: To determine the treatment schedule, the number of lymphocyte subsets in the peripheral blood of mice was traced by flow cytometry after administration of anti-CD4 mAb. The ovalbumin (OVA)257-264 peptide vaccine was injected intradermally and anti-CD4 mAb was administered intraperitoneally into C57BL/6 mice at different schedules. We evaluated the enhancement of OVA peptide-specific cytotoxic T lymphocyte (CTL) induction in the combination therapy using the ELISPOT assay, CD107a assay, and cytokine assay. We then examined the in vivo metastasis inhibitory effect by OVA peptide vaccine therapy in combination with anti-CD4 mAb against OVA-expressing thymoma (EG7) in a murine liver metastatic model. RESULTS: We showed that peptide-specific CTL induction was enhanced by the peptide vaccine in combination with anti-CD4 mAb and that the optimized treatment schedule had the strongest induction effect of peptide-specific CTLs using an IFN-γ ELISPOT assay. We also confirmed that the CD107a+ cells secreted perforin and granzyme B and the amount of IL-2 and TNF produced by these CTLs increased when the peptide vaccine was combined with anti-CD4 mAb. Furthermore, metastasis was inhibited by peptide vaccines in combination with anti-CD4 mAb compared to peptide vaccine alone in a murine liver metastatic model. CONCLUSION: The use of anti-CD4 mAb in combination with the OVA peptide vaccine therapy increased the number of peptide-specific CTLs and showed a higher therapeutic effect against OVA-expressing tumors. The combination with anti-CD4 mAb may provide a new cancer vaccine strategy.