Circulating cancer-associated extracellular vesicles as early detection and recurrence biomarkers for pancreatic cancer.

Early detection of pancreatic ductal adenocarcinoma (PDAC) is essential for improving patient survival rates, and noninvasive biomarkers are urgently required to identify patients who are eligible for curative surgery. Here, we examined extracellular vesicles (EVs) from the serum of PDAC patients to determine their ability to detect early-stage disease. EV-associated proteins purified by ultracentrifugation and affinity columns underwent proteomic analysis to identify novel PDAC markers G protein-coupled receptor class C group 5 member C (GPRC5C) and epidermal growth factor receptor pathway substrate 8 (EPS8). To verify the potency of GPRC5C- or EPS8-positive EVs as PDAC biomarkers, we analyzed EVs from PDAC patient blood samples using ultracentrifugation in two different cohorts (a total of 54 PDAC patients, 32 healthy donors, and 22 pancreatitis patients) by immunoblotting. The combination of EV-associated GPRC5C and EPS8 had high accuracy, with area under the curve values of 0.922 and 0.946 for distinguishing early-stage PDAC patients from healthy controls in the two cohorts, respectively, and could detect PDAC patients who were negative for CA19-9. Moreover, we analyzed 30 samples taken at three time points from 10 PDAC patients who underwent surgery: before surgery, after surgery, and recurrence as an early-stage model. These proteins were detected in EVs derived from preoperative and recurrence samples. These results indicated that GPRC5C- or EPS8-positive EVs were biomarkers that have the potential to detect stage I early pancreatic cancer and small recurrent tumors detected by computed tomography.

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.

Heat Shock Protein 105 as an Immunotherapeutic Target for Patients With Cervical Cancer.

BACKGROUND/AIM: Heat shock protein 105 (HSP105) is overexpressed in various cancers, but not in normal tissues. We investigated the expression levels of HSP105 in cervical cancer and the efficacy of immunotherapy targeting HSP105. MATERIALS AND METHODS: Previously, we established human leukocyte antigen-A*02:01 (HLA-A2) restricted HSP105 peptide-specific cytotoxic T lymphocyte (CTL) clones from a colorectal cancer patient vaccinated with an HSP105 peptide. Herein, we evaluated the expression of HSP105 in cervical cancer and cervical intraepithelial neoplasia. Moreover, we tested the effectiveness of an HLA-A2-restricted HSP105 peptide-specific CTL clone against cervical cancer cell lines. RESULTS: HSP105 was expressed in 95% (19/20) of examined cervical cancer tissues. Moreover, the HSP105 peptide-specific CTL clone recognized HSP105- and HLA-A*02:01-positive cervical cancer cell lines and also showed that cytotoxicity against the cervical cancer cell lines depends on HSP105 peptide and HLA class I restricted manners. CONCLUSION: HSP105 could be an effective target for immunotherapy in patients with cervical 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.

Greater extent of blood-tumor TCR repertoire overlap is associated with favorable clinical responses to PD-1 blockade.

With the widespread use of programmed death receptor-1 (PD-1) blockade therapy, sensitive and specific predictive biomarkers that guide patient selection are urgently needed. T-cell receptor (TCR) repertoire, which reflects antitumor T-cell responses based on antigen specificity, is expected as a novel biomarker for PD-1 blockade therapy. In the present study, the TCR repertoire of eight patients with gastrointestinal cancer treated with anti-PD-1 antibody (nivolumab) was analyzed. To analyze the tumor-associated T-cell clones in the blood and their mobilization into the tumor, we focused on T-cell clones that presented in both blood and tumor (blood-tumor overlapping clones). Responders to PD-1 blockade tended to exhibit a higher number of overlapping clones in the tumor and a higher total frequency in the blood. Moreover, a higher total frequency of overlapping clones in blood CD8+ T cells before treatment was associated with a favorable clinical response. Collectively, these results suggest the possibility of blood-tumor TCR repertoire overlap to predict clinical response to PD-1 blockade and guide patient selection before the treatment.

Transient Depletion of CD4+ Cells Induces Remodeling of the TCR Repertoire in Gastrointestinal Cancer.

Antibody-mediated transient depletion of CD4+ cells enhances the expansion of tumor-reactive CD8+ T cells and exhibits robust antitumor effects in preclinical and clinical studies. To investigate the clonal T-cell responses following transient CD4+ cell depletion in patients with cancer, we conducted a temporal analysis of the T-cell receptor (TCR) repertoire in the first-in-human clinical trial of IT1208, a defucosylated humanized monoclonal anti-CD4. Transient depletion of CD4+ cells promoted replacement of T-cell clones among CD4+ and CD8+ T cells in the blood. This replacement of the TCR repertoire was associated with the extent of CD4+ T-cell depletion and an increase in CD8+ T-cell count in the blood. Next, we focused on T-cell clones overlapping between the blood and tumor in order to track tumor-associated T-cell clones in the blood. The total frequency of blood-tumor overlapping clones tended to increase in patients receiving a depleting dose of anti-CD4, which was accompanied by the replacement of overlapping clones. The greater expansion of CD8+ overlapping clones was commonly observed in the patients who achieved tumor shrinkage. These results suggested that the clonal replacement of the TCR repertoire induced by transient CD4+ cell depletion was accompanied by the expansion of tumor-reactive T-cell clones that mediated antitumor responses. Our findings propose beneficial remodeling of the TCR repertoire following transient CD4+ cell depletion and provide novel insight into the antitumor effects of monoclonal anti-CD4 treatment in patients with cancer.See related Spotlight on p. 601.

Efficacy of immunotherapy targeting the neoantigen derived from epidermal growth factor receptor T790M/C797S mutation in non-small cell lung cancer.

Lung cancer is the leading cause of cancer-related deaths worldwide. Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI) often have good clinical activity against non-small cell lung cancer (NSCLC) with activating EGFR mutations. Osimertinib, which is a third-generation EGFR-TKI, has a clinical effect even on NSCLC harboring the threonine to methionine change at codon 790 of EGFR (EGFR T790M) mutation that causes TKI resistance. However, most NSCLC patients develop acquired resistance to osimertinib within approximately 1 year, and 40% of these patients have the EGFR T790M and cysteine to serine change at codon 797 (C797S) mutations. Therefore, there is an urgent need for the development of novel treatment strategies for NSCLC patients with the EGFR T790M/C797S mutation. In this study, we identified the EGFR T790M/C797S mutation-derived peptide (790-799) (MQLMPFGSLL) that binds the human leukocyte antigen (HLA)-A*02:01, and successfully established EGFR T790M/C797S-peptide-specific CTL clones from human PBMC of HLA-A2 healthy donors. One established CTL clone demonstrated adequate cytotoxicity against T2 cells pulsed with the EGFR T790M/C797S peptide. This CTL clone also had high reactivity against cancer cells that expressed an endogenous EGFR T790M/C797S peptide using an interferon-γ (IFN-γ) enzyme-linked immunospot (ELISPOT) assay. In addition, we demonstrated using a mouse model that EGFR T790M/C797S peptide-specific CTL were induced by EGFR T790M/C797S peptide vaccine in vivo. These findings suggest that an immunotherapy targeting a neoantigen derived from EGFR T790M/C797S mutation could be a useful novel therapeutic strategy for NSCLC patients with EGFR-TKI resistance, especially those resistant to osimertinib.

Ki67 expression and localization of T cells after neoadjuvant therapies as reliable predictive markers in rectal cancer.

Chemoradiotherapy (CRT) is the standard neoadjuvant therapy for locally advanced rectal cancer (RC). However, neoadjuvant chemotherapy (NAC) also shows favorable outcomes. Although the immunological environment of RC has been thoroughly discussed, the effect of NAC on it is less clear. Here, we investigated the immunological microenvironment, including T cell infiltration, activation, and topological distribution, of resected RC tissue after neoadjuvant therapies and evaluated the correlation between T cell subsets and patient prognosis. Rectal cancer patients (n = 188) were enrolled and categorized into 3 groups, namely CRT (n = 41), NAC (n = 46), and control (surgery alone; n = 101) groups. Characterization of residual carcinoma cells and T cell subsets in resected tissues was performed using multiplex fluorescence immunohistochemistry. The densities of total and activated (Ki67high ) T cells in tissues after NAC, but not CRT, were higher than in control. In both CRT and NAC groups, patients presenting with higher treatment effects showed aggressive infiltration of T cell subsets into carcinomas. Multivariate analyses of pathological and immunological features and prognosis revealed that carcinoma Ki67high CD4+ T cells after CRT and stromal Ki67high CD8+ T cells after NAC are important prognostic factors, respectively. Our results suggest that evaluation of T cell activation with Ki67 expression and its tumor localization can be used to determine the prognosis of advanced RC after neoadjuvant therapies.

Efficacy of the NCCV Cocktail-1 vaccine for refractory pediatric solid tumors: A phase I clinical trial.

Pediatric refractory solid tumors are aggressive malignant diseases, resulting in an extremely poor prognosis. KOC1, FOXM1, and KIF20A are cancer antigens that could be ideal targets for anticancer immunotherapy against pediatric refractory solid tumors with positive expression for these antigens. This nonrandomized, open-label, phase I clinical trial evaluated the safety and efficacy of the NCCV Cocktail-1 vaccine, which is a cocktail of cancer peptides derived from KOC1, FOXM1, and KIF20A, in patients with pediatric refractory solid tumors. Twelve patients with refractory pediatric solid tumors underwent NCCV Cocktail-1 vaccination weekly by intradermal injections. The primary endpoint was the safety of the NCCV Cocktail-1 vaccination, and the secondary endpoints were the immune response, as measured by interferon-r enzyme-linked immunospot assay, and the clinical outcomes including tumor response and progression-free survival. The NCCV Cocktail-1 vaccine was well tolerated. The clinical response of this trial showed that 4 patients had stable disease after 8 weeks and 2 patients maintained remission for >11 months. In 4, 8, and 5 patients, the NCCV Cocktail-1 vaccine induced the sufficient number of peptide-specific CTLs for KOC1, FOXM1, and KIF20A, respectively. Patients with high peptide-specific CTL frequencies for KOC1, FOXM1, and KIF20A had better progression-free survival than those with low frequencies. The findings of this clinical trial showed that the NCCV Cocktail-1 vaccine could be a novel therapeutic strategy, with adequate effects against pediatric refractory solid tumors. Future large-scale trials should evaluate the efficacy of the NCCV Cocktail-1 vaccination.

First-in-human phase 1 study of IT1208, a defucosylated humanized anti-CD4 depleting antibody, in patients with advanced solid tumors.

BACKGROUND: Transient CD4+ T cell depletion led to the proliferation of tumor-specific CD8+ T cells in the draining lymph node and increased infiltration of PD-1+CD8+ T cells into the tumor, which resulted in strong anti-tumor effects in tumor-bearing mice. This is a first-in-human study of IT1208, a defucosylated humanized anti-CD4 monoclonal antibody, engineered to exert potent antibody-dependent cellular cytotoxicity. METHODS: Patients with advanced solid tumors were treated with intravenous IT1208 at doses of 0.1 or 1.0 mg/kg. The first patient in each cohort received a single administration, and the other patients received two administrations of IT1208 on days 1 and 8. RESULTS: Eleven patients were enrolled in the 0.1 mg/kg (n = 4) and 1.0 mg/kg cohorts (n = 7). Grade 1 or 2 infusion-related reactions was observed in all patients. Decreased CD4+ T cells in peripheral blood due to IT1208 were observed in all patients and especially in those receiving two administrations of 1.0 mg/kg. CD8+ T cells increased on day 29 compared with baseline in most patients, resulting in remarkably decreased CD4/8 ratios. One microsatellite-stable colon cancer patient achieved durable partial response showing increased infiltration of both CD4+ and CD8+ T cells into tumors after IT1208 administration. Moreover, transcriptomic profiling of the liver metastasis of the patient revealed upregulation of the expression of interferon-stimulated genes, T cell activation-related genes, and antigen presentation-related genes after IT1208 administration. Two additional patients with gastric or esophageal cancer achieved stable disease lasting at least 3 months. CONCLUSIONS: IT1208 monotherapy successfully depleted CD4+ T cells with a manageable safety profile and encouraging preliminary efficacy signals, which warrants further investigations, especially in combination with immune checkpoint inhibitors.

Higher human lymphocyte antigen class I expression in early-stage cancer cells leads to high sensitivity for cytotoxic T lymphocytes.

Human lymphocyte antigen (HLA) class I molecules play a central role in cytotoxic T lymphocytes (CTL)-based antitumor immunity. However, the expression rate of HLA class I in cancer cells remains a topic of discussion. We compared HLA class I expression levels between cancer cells and surrounding non-tumorous hepatocytes in 20 early-stage hepatocellular carcinoma (HCC) patients by immunohistochemistry using EMR 8-5. The expression levels of HLA class I were classified as negative, incomplete positive or complete positive. Similarly, for various types of solid cancers, HLA class I expression was examined. For the HLA class I expression in cancer cells, among 20 HCC patients, 13 were complete positive, 3 were incomplete positive, and 4 were negative. In addition, 15 (75.0%) had higher expression levels of HLA class I in cancer cells compared with that in surrounding non-tumorous hepatocytes. An interferon-γ (IFN-γ) enzyme-linked immunospot (ELISPOT) assay indicated that cancer cells with positive expression of HLA class I had strong sensitivity to antigen-specific CTL. We suggested that HLA class I expression in cancer cells could be involved in the clinical prognosis of HCC patients. Similarly, 66.7%, 100.0%, 66.7% and 62.5% of patients with early-stage pancreatic, gallbladder, esophageal and breast cancers, respectively, had higher expression levels of HLA class I in cancer cells than in surrounding normal tissue cells. We suggest that in several early-stage solid cancers, including HCC, HLA class I expression levels in cancer cells are higher than that in surrounding normal tissue cells, which could result in the anti-tumor effect of CTL-based cancer immunotherapy.

Selective elimination of undifferentiated human pluripotent stem cells using pluripotent state-specific immunogenic antigen Glypican-3.

Immunogenicity of immature pluripotent stem cells is a topic of intense debate. Immunogenic antigens, which are specific in pluripotent states, have not been described previously. In this study, we identified glypican-3 (GPC3), a known carcinoembryonic antigen, as a pluripotent state-specific immunogenic antigen. Additionally, we validated the applicability of human leukocyte antigen (HLA)-class I-restricted GPC3-reactive cytotoxic T lymphocytes (CTLs) in the removal of undifferentiated pluripotent stem cells (PSCs) from human induced pluripotent stem cell (hiPSC)-derivatives. HiPSCs uniquely express GPC3 in pluripotent states and were rejected by GPC3-reactive CTLs, which were sensitized with HLA-class I-restricted GPC3 peptides. Furthermore, GPC3-reactive CTLs selectively removed undifferentiated PSCs from hiPSC-derivatives in vitro and inhibited tumor formation in vivo. Our results demonstrate that GPC3 works as a pluripotent state-specific immunogenic antigen in hiPSCs and is applicable to regenerative medicine as a method of removing undifferentiated PSCs, which are the main cause of tumor formation.

Organoids with cancer stem cell-like properties secrete exosomes and HSP90 in a 3D nanoenvironment.

Ability to form cellular aggregations such as tumorspheres and spheroids have been used as a morphological marker of malignant cancer cells and in particular cancer stem cells (CSC). However, the common definition of the types of cellular aggregation formed by cancer cells has not been available. We examined morphologies of 67 cell lines cultured on three dimensional morphology enhancing NanoCulture Plates (NCP) and classified the types of cellular aggregates that form. Among the 67 cell lines, 49 cell lines formed spheres or spheroids, 8 cell lines formed grape-like aggregation (GLA), 8 cell lines formed other types of aggregation, and 3 cell lines formed monolayer sheets. Seven GLA-forming cell lines were derived from adenocarcinoma among the 8 lines. A neuroendocrine adenocarcinoma cell line PC-3 formed asymmetric GLA with ductal structures on the NCPs and rapidly growing asymmetric tumors that metastasized to lymph nodes in immunocompromised mice. In contrast, another adenocarcinoma cell line DU-145 formed spheroids in vitro and spheroid-like tumors in vivo that did not metastasize to lymph nodes until day 50 after transplantation. Culture in the 3D nanoenvironment and in a defined stem cell medium enabled the neuroendocrine adenocarcinoma cells to form slowly growing large organoids that expressed multiple stem cell markers, neuroendocrine markers, intercellular adhesion molecules, and oncogenes in vitro. In contrast, the more commonly used 2D serum-contained environment reduced intercellular adhesion and induced mesenchymal transition and promoted rapid growth of the cells. In addition, the 3D stemness nanoenvironment promoted secretion of HSP90 and EpCAM-exosomes, a marker of CSC phenotype, from the neuroendocrine organoids. These findings indicate that the NCP-based 3D environment enables cells to form stem cell tumoroids with multipotency and model more accurately the in vivo tumor status at the levels of morphology and gene expression.

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.

Perioperative plasma glypican-3 level may enable prediction of the risk of recurrence after surgery in patients with stage I hepatocellular carcinoma.

Glypican-3 (GPC3) is a glycosylphosphatidylinositol-anchored cell surface protein overexpressed in hepatocellular carcinoma(HCC), and its overexpression is associated with poor prognosis. The diagnostic potential of GPC3 as a serum marker has been reported. In the present study, we evaluated the usefulness of plasma GPC3 as a predictor for recurrence after surgical resection in stage I HCC patients by newly developed an enzyme-linked immunosorbent assay (ELISA) system. Current study demonstrated that high levels of preoperative plasma GPC3 patients tended to experience postoperative recurrence. On the other hand, pre- and postoperative plasma GPC3 positivity of non-recurrence patients was very low. Moreover, even after surgery, approximately half of patients who experienced recurrence were positive for plasma GPC3. Postoperative plasma GPC3 positivity was significantly correlated with worse recurrence-free survival. Immuohistochemical analysis also showed positive rate of GPC3-expression in HCC was higher in recurrence patients than in non-recurrence patients. These results suggested that both pre- and postoperative plasma GPC3 levels may be accurate predictors for recurrence after curative resection of early-stage HCC. It should be noted that the current study only examined a small number of cases; thus, a larger sample size is necessary to validate GPC3 as a predictor for HCC recurrence.

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.

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.

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.

Time-lapse imaging assay using the BioStation CT: a sensitive drug-screening method for three-dimensional cell culture.

Three-dimensional (3D) cell culture is beneficial for physiological studies of tumor cells, due to its potential to deliver a high quantity of cell culture information that is representative of the cancer microenvironment and predictive of drug responses in vivo. Currently, gel-associated or matrix-associated 3D cell culture is comprised of intricate procedures that often result in experimental complexity. Therefore, we developed an innovative anti-cancer drug sensitivity screening technique for 3D cell culture on NanoCulture Plates (NCP) by employing the imaging device BioStation CT. Here, we showed that the human breast cancer cell lines BT474 and T47D form multicellular spheroids on NCP plates and compared their sensitivity to the anti-cancer drugs trastuzumab and paclitaxel using the BioStation CT. The anticancer drugs reduced spheroid migration velocity and suppressed spheroid fusion. In addition, primary cells derived from the human breast cancer tissues B58 and B61 grown on NCP plates also exhibited similar drug sensitivity. These results were in good agreement with the conventional assay method using ATP quantification. We confirmed the antitumor effects of the drugs on cells seeded in 96-well plates using the BioStation CT imaging technique. We expect this method to be useful in research for new antitumor agents and for drug sensitivity tests in individually-tailored cancer treatments.

Programmed death-1 blockade enhances the antitumor effects of peptide vaccine-induced peptide-specific cytotoxic T lymphocytes.

Novel treatment modalities are required urgently in patients with hepatocellular carcinoma (HCC). A vaccine that induces cytotoxic T lymphocytes (CTLs) is an ideal strategy for cancer, and glypican-3 (GPC3) is a potential option for HCC. Blocking the programmed death-1 (PD-1)/PD-L1 pathway is a rational strategy to overcome tumor escape and tolerance toward CTLs. In the present study, we investigated whether anti-PD-1 blocking antibodies (αPD-1 Ab) enhanced the number of vaccine-induced peptide-specific CTLs in peripheral blood mononuclear cells (PBMCs) following the administration of GPC3 peptide vaccine to both patients and in a mouse model. The inhibitory receptor PD-1 was highly expressed in ex vivo GPC3-specific CTLs isolated from the PBMCs of vaccinated HCC patients. In vitro, interferon-γ induced PD-L1 expression in liver cancer cell lines. In addition, PD-1 blockade increased the number of GPC3-specific CTLs, which degranulate against liver cancer cell lines. In vivo experiments using tumor-bearing mouse models showed that the combination therapy of peptide vaccine and αPD-1 Ab suppressed tumor growth synergistically. PD-1 blockade increased the number of peptide-specific tumor-infiltrating T cells (TILs) and decreased the expression of inhibitory receptors on TILs. This study demonstrated that PD-1/PD-L1 blockade augmented the antitumor effects of a peptide vaccine by increasing the immune response of vaccine-induced CTLs, and provided a foundation for the clinical development of a combination therapy using a GPC3 peptide vaccine and αPD-1 Ab.