Tetsuya Nakatsura

Glypican-3, overexpressed specifically in human hepatocellular carcinoma, is a novel tumor marker

With the global pandemic of hepatitis B and C infections, the incidence of Hepatocellular carcinoma (HCC) is rapidly increasing world wide. We identified glypican-3 (GPC3), a novel oncofetal gene over-expressed specifically in human HCC, as based on data of cDNA microarrays. As GPC3 is a GPI-anchored membrane protein and could be secreted, we attempted to detect secreted GPC3 protein in sera from HCC patients using Western blotting and ELISA. GPC3 protein was positive in sera of 40.0% (16/40) of HCC patients, and negative in sera from subjects with liver cirrhosis (LC) (0/13), chronic hepatitis (CH) (0/34), and healthy donors (0/60). All subjects were Japanese. Although 12 of 40 HCC patients were negative for both alpha-fetoprotein (AFP) and PIVKA-II well known tumor markers of HCC, four of these were GPC3-positive in the sera. We also observed vanishing GPC3 protein in the sera of three patients after the surgical treatment for HCC. On the other hand, immunohistochemical analysis revealed that HCC expressed GPC3 protein in all 14 HCC patients tested. In conclusion, GPC3, as defined in this study was shown to be a useful tumor marker for cancer-diagnosis for large numbers of patients with HCC.

Identification of HLA-A2- or HLA-A24-Restricted CTL Epitopes Possibly Useful for Glypican-3-Specific Immunotherapy of Hepatocellular Carcinoma

Purpose and Experimental Design: We previously reported that glypican-3 (GPC3) was overexpressed, specifically in hepatocellular carcinoma (HCC) and melanoma in humans, and it was useful as a novel tumor marker. We also reported that the preimmunization of BALB/c mice with dendritic cells pulsed with the H-2Kd-restricted mouse GPC3298-306 (EYILSLEEL) peptide prevented the growth of tumor-expressing mouse GPC3. Because of similarities in the peptide binding motifs between H-2Kd and HLA-A24 (A*2402), the GPC3298-306 peptide therefore seemed to be useful for the immunotherapy of HLA-A24+ patients with HCC and melanoma. In this report, we investigated whether the GPC3298-306 peptide could induce GPC3-reactive CTLs from the peripheral blood mononuclear cells (PBMC) of HLA-A24 (A*2402)+ HCC patients. In addition, we used HLA-A2.1 (HHD) transgenic mice to identify the HLA-A2 (A*0201)–restricted GPC3 epitopes to expand the applications of GPC3-based immunotherapy to the HLA-A2+ HCC patients. Results: We found that the GPC3144-152 (FVGEFFTDV) peptide could induce peptide-reactive CTLs in HLA-A2.1 (HHD) transgenic mice without inducing autoimmunity. In five out of eight HLA-A2+ GPC3+ HCC patients, the GPC3144-152 peptide-reactive CTLs were generated from PBMCs by in vitro stimulation with the peptide and the GPC3298-306 peptide-reactive CTLs were also generated from PBMCs in four of six HLA-A24+ GPC3+ HCC patients. The inoculation of these CTLs reduced the human HCC tumor mass implanted into nonobese diabetic/severe combined immunodeficiency mice. Conclusion: Our study raises the possibility that these GPC3 peptides may therefore be applicable to cancer immunotherapy for a large number of HCC patients.

Identification of Glypican-3 as a Novel Tumor Marker for Melanoma

Purpose: We reported recently the novel tumor marker glypican-3 (GPC3) for hepatocellular carcinoma. In the present study, we investigated the expression of GPC3 in human melanoma cell lines and tissues and asked whether GPC3 could be a novel tumor marker for melanoma. Experimental Design: Expression of GPC3 mRNA and protein was investigated in human melanoma cell lines and tissues using reverse transcription-PCR and immunohistochemical analysis. Secreted GPC3 protein was quantified using ELISA in culture supernatants of melanoma cell lines and in sera from 91 patients with melanoma and 28 disease-free patients after surgical removal of primary melanoma. All of the subjects were Japanese nationals. Results: In >80% of melanoma and melanocytic nevus, there was evident expression of GPC3 mRNA and protein. Furthermore, GPC3 protein was evidenced in sera of 39.6% (36 of 91) of melanoma patients but not in sera from subjects with large congenital melanocytic nevus (0 of 5) and from healthy donors (0 of 60). Twenty-seven of 36 serum GPC3-positive patients were negative for both serum 5-S-cysteinyldopa and melanoma-inhibitory activity, well-known tumor markers for melanoma. The positive rate of serum GPC3 (39.6%) was significantly higher than that of 5-S-cysteinyldopa (26.7%) and of melanoma-inhibitory activity (20.9%). Surprisingly, we detected serum GPC3 even in patients with stage 0 in situ melanoma. The positive rate of serum GPC3 at stage 0, I, and II (44.4%, 40.0%, and 47.6%) was significantly higher than that of 5-S-cysteinyldopa (0.0%, 8.0%, and 10.0%). Also observed was the disappearance of GPC3 protein in sera from 11 patients after surgical removal of the melanoma. Conclusions: GPC3 is apparently a novel tumor marker useful for the diagnosis of melanoma, especially in early stages of the disorder.

Glypican-3 expression is correlated with poor prognosis in hepatocellular carcinoma

The relationship between overexpression of glypican (GPC)‐3 that is specific for hepatocellular carcinoma (HCC) and the prognosis has not yet been clarified. We attempted to determine the expression profile of GPC3 in association with the clinicopathological factors by immunohistochemical analysis in HCC patients and investigated the potential prognostic value of GPC3 by comparing the survival rate between the GPC3‐positive and GPC3‐negative HCC patients. Primary HCC tissue samples (n = 107) obtained from patients who had undergone hepatectomy between 2000 and 2001 were analyzed. GPC3 expression was less frequently observed in well‐differentiated HCC than in moderately and poorly differentiated HCC, the difference in the frequency being statistically significant. GPC3‐positive HCC patients had a significantly lower 5‐year survival rate than the GPC3‐negative HCC patients (54.5 vs 87.7%, P = 0.031). Among 80 of the 107 (74.6%) patients with initial treatment who underwent hepatectomy, none of GPC3‐negative HCC patients (n = 16, 20.0%) died during the follow‐up period. No deaths were noted in the GPC3‐negative HCC patients among the 71 (88.7%) patients with moderately and poorly differentiated HCC. Multivariate analysis identified GPC3 expression (P = 0.034) as an independent prognostic factor for the overall survival. We showed that GPC3 expression is correlated with a poor prognosis in HCC patients. (Cancer Sci 2009)

Phase I Trial of a Glypican-3–Derived Peptide Vaccine for Advanced Hepatocellular Carcinoma: Immunologic Evidence and Potential for Improving Overall Survival

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.

Cellular and humoral immune responses to a human pancreatic cancer antigen, coactosin‐like protein, originally defined by the SEREX method

Among a number of human tumor antigens identified using the serological analysis of recombinant cDNA expression libraries (SEREX), only MAGE‐1, tyrosinase, and NY‐ESO‐1 have been reported to be immunogenic tumor antigens that have the potential to elicit both humoral and cellular immunity. In this study, we determined whether our SEREX‐defined pancreatic cancer antigens could be recognized by CTL, and report that one SEREX‐defined antigen, coactosin‐like protein (CLP), encoded cellular epitopes recognized by HLA‐A2‐restricted and tumor‐reactive CTL. Three CLP peptides at positions 15–24, 57–65, and 104–113 possessed the ability to induce HLA‐A2‐restricted and tumor‐reactive CTL from the PBMC of cancer patients. Subsequently, humoral responses to these peptides were investigated. IgG antibodies specific to the CLP 15–24, 57–65, and 104–113 peptides were detected in sera from 12, 0, and 12 of 12 cancer patients tested, and were also found in 5, 0, and 0 of 9 healthy donors, respectively. IgE antibodies specific to these peptides were also detected in sera from certain cancer patients and healthy donors. Since peptide‐specific IgE was detected, type‐I allergy to these peptides was tested. Unexpectedly the CLP 57–65 peptide, to which IgE was found in only 2 healthy donors, but not the other two peptides, was found to elicit an immediate‐type hypersensitivity in all10 healthy volunteers tested. These results indicate that identical antigenic peptides can be recognized by both cellular and humoral immune systems to a tumor‐associated antigen. The CLP 15–24 and104–113 peptides might be appropriate vaccine candidates for peptide‐based immunotherapy of HLA‐A2+ cancer patients.

Mouse Homologue of a Novel Human Oncofetal Antigen, Glypican-3, Evokes T-Cell–Mediated Tumor Rejection without Autoimmune Reactions in Mice

Purpose and Experimental Design: We recently identified glypican-3 (GPC3) overexpressed specifically in human hepatocellular carcinoma, as based on cDNA microarray analysis of 23,040 genes, and we reported that GPC3 is a novel tumor marker for human hepatocellular carcinoma and melanoma. GPC3, expressed in almost all hepatocellular carcinomas and melanomas, but not in normal tissues except for placenta or fetal liver, is a candidate of ideal tumor antigen for immunotherapy. In this study, we attempted to identify a mouse GPC3 epitope for CTLs in BALB/c mice, and for this, we set up a preclinical study to investigate the usefulness of GPC3 as a target for cancer immunotherapy in vivo. Results: We identified a mouse GPC3-derived and Kd- restricted CTL epitope peptide in BALB/c mice. Inoculation of this GPC3 peptide-specific CTL into s.c. Colon26 cancer cells transfected with mouse GPC3 gene (C26/GPC3) led to rejection of the tumor in vivo, and i.v. inoculation of these CTLs into sublethally irradiated mice markedly inhibited growth of an established s.c. tumor. Inoculation of bone marrow-derived dendritic cells pulsed with this peptide prevented the growth of s.c. and splenic C26/GPC3 accompanied with massive infiltration of CD8+ T cells into tumors. Evidence of autoimmune reactions was never observed in surviving mice that had rejected tumor cell challenges. Conclusions: We found the novel oncofetal protein GPC3 to be highly immunogenic in mice and elicited effective antitumor immunity with no evidence of autoimmunity. GPC3 is useful not only for diagnosis of hepatocellular carcinoma and melanoma but also for possible immunotherapy or prevention of these tumors.

HLA-A2-restricted CTL epitopes of a novel lung cancer-associated cancer testis antigen, cell division cycle associated 1, can induce tumor-reactive CTL

Toward the development of a novel cancer immunotherapy, we have previously identified several tumor‐associated antigens (TAAs) and the epitopes recognized by human histocompatibility leukocyte (HLA)‐A2/A24‐restricted cytotoxic T lymphocyte (CTL). In this study, we tried to identify a TAA of lung cancer (LC) and its HLA‐A2 restricted CTL epitopes to provide a target antigen useful for cancer immunotherapy of LC. We identified a novel cancer testis antigen, cell division cycle associated gene 1 (CDCA1), overexpressed in nonsmall cell LC using a cDNA microarray analysis. The expression levels of CDCA1 were also increased in the majority of small cell LC, cholangiocellular cancer, urinary bladder cancer and renal cell cancers. We used HLA‐A2.1 transgenic mice to identify the HLA‐A2 (A*0201)‐restricted CDCA1 epitopes recognized by mouse CTL, and we investigated whether these peptides could induce CDCA1‐reactive CTLs from the peripheral blood mononuclear cells (PBMCs) of HLA‐A2‐positive donors and a NSCLC patient. Consequently, we found that the CDCA165–73 (YMMPVNSEV) peptide and CDCA1351–359 (KLATAQFKI) peptide could induce peptide‐reactive CTLs in HLA‐A2.1 transgenic mice. In HLA‐A2+ donors, in vitro stimulation of PBMC with these peptides could induce peptide‐reactive CTLs which killed tumor cell lines endogenously expressing both HLA‐A2 and CDCA1. As a result, CDCA1 is a novel cancer‐testis antigen overexpressed in LC, cholangiocellular cancer, urinary bladder cancer and renal cell cancers, and CDCA1 may therefore be an ideal TAA useful for the diagnosis and immunotherapy of these cancers.

Embryonic Stem Cell–Derived Dendritic Cells Expressing Glypican-3, a Recently Identified Oncofetal Antigen, Induce Protective Immunity against Highly Metastatic Mouse Melanoma, B16-F10

We have recently established a method to generate dendritic cells from mouse embryonic stem cells. By introducing exogenous genes into embryonic stem cells and subsequently inducing differentiation to dendritic cells (ES-DC), we can now readily generate transfectant ES-DC expressing the transgenes. A previous study revealed that the transfer of genetically modified ES-DC expressing a model antigen, ovalbumin, protected the recipient mice from a challenge with an ovalbumin-expressing tumor. In the present study, we examined the capacity of ES-DC expressing mouse homologue of human glypican-3, a recently identified oncofetal antigen expressed in human melanoma and hepatocellular carcinoma, to elicit protective immunity against glypican-3-expressing mouse tumors. CTLs specific to multiple glypican-3 epitopes were primed by the in vivo transfer of glypican-3-transfectant ES-DC (ES-DC-GPC3). The transfer of ES-DC-GPC3 protected the recipient mice from subsequent challenge with B16-F10 melanoma, naturally expressing glypican-3, and with glypican-3-transfectant MCA205 sarcoma. The treatment with ES-DC-GPC3 was also highly effective against i.v. injected B16-F10. No harmful side effects, such as autoimmunity, were observed for these treatments. The depletion experiments and immunohistochemical analyses suggest that both CD8+ and CD4+ T cells contributed to the observed antitumor effect. In conclusion, the usefulness of glypican-3 as a target antigen for antimelanoma immunotherapy was thus shown in the mouse model using the ES-DC system. Human dendritic cells expressing glypican-3 would be a promising means for therapy of melanoma and hepatocellular carcinoma.

Proliferation Potential-Related Protein, an Ideal Esophageal Cancer Antigen for Immunotherapy, Identified Using Complementary DNA Microarray Analysis

Purpose: To establish effective antitumor immunotherapy for esophageal cancer, we tried to identify an useful target antigen of esophageal cancer. Experimental Design: We did cDNA microarray analysis to find a novel candidate antigen, proliferation potential-related protein (PP-RP). We examined cytotoxicity against tumor cells in vitro and in vivo of CTLs specific to PP-RP established from esophageal cancer patients. Results: In 26 esophageal cancer tissues, an average of relative ratio of the expression of the PP-RP mRNA in cancer cells versus adjacent normal esophageal tissues was 396.2. Immunohistochemical analysis revealed that, in 20 of the 22 esophageal cancer tissues, PP-RP protein was strongly expressed only in the cancer cells and not so in normal esophageal epithelial cells. PP-RP protein contains 10 epitopes recognized by HLA-A24–restricted CTLs. These CTLs, generated from HLA-A24–positive esophageal cancer patients, had cytotoxic activity against cancer cell lines positive for both PP-RP and HLA-A24. Furthermore, adoptive transfer of the PP-RP–specific CTL line inhibited the growth of a human esophageal cancer cell line engrafted in nude mice. Conclusions: The expression of PP-RP in esophageal cancer cells was significantly higher than in normal cells, and the CTLs recognizing PP-RP killed tumor cells in vitro and also showed tumor rejection effects in a xenograft model. Therefore, PP-RP may prove to be an ideal tumor antigen useful for diagnosis and immunotherapy for patients with esophageal cancer. cDNA microarray analysis is a useful method to identify ideal tumor-associated antigens.