Satoru Senju

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.

Ribosome stalling induced by mutation of a CNS-specific tRNA causes neurodegeneration

Problems making proteins kills nerve cells Neurodegeneration is associated with a variety of different diseases, but its cellular roots are often obscure. Ishimura et al. find that mutant mice whose brain cells start to die rapidly soon after birth have lost the function of two vital cellular components (see the Perspective by Darnell). The first is a protein that releases stalled ribosomes stuck on messenger RNA (mRNA); the second is a transfer RNA (tRNA), which reads the code for arginine in the mRNA. This tRNA is expressed predominantly in the central nervous system. The lack of the tRNA leads to increased ribosomal stalling at arginine codons, which, when left uncorrected, blocks protein synthesis and proves fatal. Science, this issue p. 455; see also p. 378 Mutations in a transfer RNA expressed in the nervous system stall ribosomes and can cause cell death if ribosome recycling fails. [Also see Perspective by Darnell] In higher eukaryotes, transfer RNAs (tRNAs) with the same anticodon are encoded by multiple nuclear genes, and little is known about how mutations in these genes affect translation and cellular homeostasis. Similarly, the surveillance systems that respond to such defects in higher eukaryotes are not clear. Here, we discover that loss of GTPBP2, a novel binding partner of the ribosome recycling protein Pelota, in mice with a mutation in a tRNA gene that is specifically expressed in the central nervous system causes ribosome stalling and widespread neurodegeneration. Our results not only define GTPBP2 as a ribosome rescue factor but also unmask the disease potential of mutations in nuclear-encoded tRNA genes.

Prevention of Experimental Autoimmune Encephalomyelitis by Transfer of Embryonic Stem Cell-Derived Dendritic Cells Expressing Myelin Oligodendrocyte Glycoprotein Peptide along with TRAIL or Programmed Death-1 Ligand

Experimental autoimmune encephalomyelitis (EAE) is caused by activation of myelin Ag-reactive CD4+ T cells. In the current study, we tested a strategy to prevent EAE by pretreatment of mice with genetically modified dendritic cells (DC) presenting myelin oligodendrocyte glycoprotein (MOG) peptide in the context of MHC class II molecules and simultaneously expressing TRAIL or Programmed Death-1 ligand (PD-L1). For genetic modification of DC, we used a recently established method to generate DC from mouse embryonic stem cells (ES cells) in vitro (ES-DC). ES cells were sequentially transfected with an expression vector for TRAIL or PD-L1 and an MHC class II-associated invariant chain-based MOG epitope-presenting vector. Subsequently, double-transfectant ES cell clones were induced to differentiate to ES-DC, which expressed the products of introduced genes. Treatment of mice with either of the double-transfectant ES-DC significantly reduced T cell response to MOG, cell infiltration into spinal cord, and the severity of MOG peptide-induced EAE. In contrast, treatment with ES-DC expressing MOG alone, irrelevant Ag (OVA) plus TRAIL, or OVA plus PD-L1, or coinjection with ES-DC expressing MOG plus ES-DC-expressing TRAIL or PD-L1 had no effect in reducing the disease severity. In contrast, immune response to irrelevant exogenous Ag (keyhole limpet hemocyanin) was not impaired by treatment with any of the genetically modified ES-DC. The double-transfectant ES-DC presenting Ag and simultaneously expressing immune-suppressive molecules may well prove to be an effective therapy for autoimmune diseases without inhibition of the immune response to irrelevant Ag.

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.

Characterization of dendritic cells and macrophages generated by directed differentiation from mouse induced pluripotent stem cells.

Methods have been established to generate dendritic cells (DCs) from mouse and human embryonic stem (ES) cells. We designated them as ES‐DCs and mouse models have demonstrated the induction of anti‐cancer immunity and prevention of autoimmune disease by in vivo administration of genetically engineered ES‐DCs. For the future clinical application of ES‐DCs, the histoincompatibility between patients to be treated and available human ES cells and the ethical concerns associated with human ES cells may be serious obstacles. However, recently developed induced pluripotent stem (iPS) cell technology is expected to resolve these issues. This report describes the generation and characterization of DCs derived from mouse iPS cells. The iPS cell‐derived DCs (iPS‐DCs) possessed the characteristics of DCs including the capacity of T‐cell‐stimulation, antigen‐processing and presentation and cytokine production. DNA microarray analyses revealed the upregulation of genes related to antigen‐presenting functions during differentiation into iPS‐DCs and similarity in gene expression profile in iPS‐DCs and bone marrow cell‐derived DCs. Genetically modified iPS‐DCs expressing antigenic protein primed T‐cells specific to the antigen in vivo and elicited efficient antigen‐specific anti‐tumor immunity. In addition, macrophages were generated from iPS cells (iPS‐MP). iPS‐MP were comparable with bone marrow cell‐derived macrophages in the cell surface phenotype, functions, and gene expression profiles. Stem Cells 2009;27:1021–1031

Identification of a Novel Tumor-Associated Antigen, Cadherin 3/P-Cadherin, as a Possible Target for Immunotherapy of Pancreatic, Gastric, and Colorectal Cancers

Purpose: To establish cancer immunotherapy, it is important to identify the tumor-associated antigens (TAA) that are strongly expressed in the tumor cells but not in the normal cells. In this study, to establish an effective anticancer immunotherapy, we tried to identify the useful TAA of pancreatic cancer. Experimental Design: Based on a previous genome-wide cDNA microarray analysis of pancreatic cancer, we focused on cadherin 3 (CDH3)/P-cadherin as a novel candidate TAA for anticancer immunotherapy. To identify the HLA-A2 (A*0201)–restricted CTL epitopes of CDH3, we used HLA-A2.1 (HHD) transgenic mice (Tgm). Furthermore, we examined the cytotoxicity against the tumor cells in vitro and in vivo of CTLs specific to CDH3 induced from HLA-A2–positive healthy donors and cancer patients. Results:CDH3 was overexpressed in the majority of pancreatic cancer and various other malignancies, including gastric and colorectal cancers, but not in their noncancerous counterparts or in many normal adult tissues. In the experiment using HLA-A2.1 Tgm, we found that the CDH3-4655-663 (FILPVLGAV) and CDH3-7757-765 (FIIENLKAA) peptides could induce HLA-A2–restricted CTLs in Tgm. In addition, peptides-reactive CTLs were successfully induced from peripheral blood mononuclear cells by in vitro stimulation with these two peptides in HLA-A2–positive healthy donors and cancer patients, and these CTLs exhibited cytotoxicity specific to cancer cells expressing both CDH3 and HLA-A2. Furthermore, the adoptive transfer of the CDH3-specific CTLs could inhibit the tumor growth of human cancer cells engrafted into nonobese diabetic/severe combined immunodeficiency mice. Conclusions: These results suggest that CDH3 is a novel TAA useful for immunotherapy against a broad spectrum of cancers, including pancreatic cancer.

Generation of dendritic cells and macrophages from human induced pluripotent stem cells aiming at cell therapy

This report describes generation of dendritic cells (DCs) and macrophages from human induced pluripotent stem (iPS) cells. iPS cell-derived DC (iPS-DC) exhibited the morphology of typical DC and function of T-cell stimulation and antigen presentation. iPS-DC loaded with cytomegalovirus (CMV) peptide induced vigorous expansion of CMV-specific autologous CD8+ T cells. Macrophages (iPS-MP) with activity of zymosan phagocytosis and C5a-induced chemotaxis were also generated from iPS cells. Genetically modified iPS-MPs were generated by the introduction of expression vectors into undifferentiated iPS cells, isolation of transfectant iPS cell clone and subsequent differentiation. By this procedure, we generated iPS-MP expressing a membrane-bound form of single chain antibody (scFv) specific to amyloid β (Aβ), the causal protein of Alzheimer’s disease. The scFv-transfectant iPS-MP exhibited efficient Aβ-specific phagocytosis activity. iPS-MP expressing CD20-specific scFv engulfed and killed BALL-1 B-cell leukemia cells. Anti-BALL-1 effect of iPS-MP in vivo was demonstrated in a xeno-transplantation model using severe combined immunodeficient mice. In addition, we established a xeno-free culture protocol to generate iPS-DC and iPS-MP. Collectively, we demonstrated the possibility of application of iPS-DC and macrophages to cell therapy.

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.