Naotake Tsuda

Identification of a Gene Coding for a New Squamous Cell Carcinoma Antigen Recognized by the CTL

Peptide-based specific immunotherapy has resulted in tumor regression in some melanoma patients. However, tumor Ags and peptides for specific immunotherapy, except for treatment of melanomas, have not yet been well identified. In this study, we report a gene encoding a new squamous cell carcinoma (SCC) Ag recognized by cells of the HLA-A24-restricted and tumor-specific CTL line. This gene with 3958-bp length was transcribed from the chromosome 6q22 with six exons, and its mRNA was ubiquitously expressed in both SCCs and normal tissues, and partly expressed in adenocarcinomas. The deduced 958-aa sequence encoded by this gene showed no similarity to any known amino acid sequences. This gene product had a characteristic of an endoplasmic reticulum-resident protein. A 100-kDa protein was detected in the vast majority of SCCs from various tissues, in majority of renal cell carcinomas and brain tumors, and in about one-third of melanomas and adenocarcinomas from various organs other than the breast. In contrast, it was not expressed at all in any of the normal cells or tissues tested, including the testis and fetal liver. Three different peptides at positions 93–101, 161–169, and 899–907 of this Ag were recognized by this CTL line, and all of them induced HLA-A24-restricted and tumor-specific CTLs from PBMCs of SCC patients. Therefore, these peptides may be useful for peptide-based specific immunotherapy of HLA-A24+ patients with SCC in various organs, as well as for treatment of other cancer.

Humoral Responses to Peptides Correlate with Overall Survival in Advanced Cancer Patients Vaccinated with Peptides Based on Pre-existing, Peptide-Specific Cellular Responses

Purpose: The aim of this study is to find a laboratory marker for overall survival in advanced cancer patients who were vaccinated with peptides based on pre-existing, peptide-specific CTL precursors in the circulation. Experimental Design: A group of 113 patients with advanced cancer (28 colorectal, 22 prostate, 15 lung, 14 gastric, and 34 other cancers) was enrolled in a Phase I clinical study of peptide vaccination in which peptide-specific CTL precursors of prevaccination peripheral blood mononuclear cells were measured, followed by vaccination with these peptides (maximum of four). For cellular responses, pre and postvaccination (sixth) peripheral blood mononuclear cells were provided for measurement of both peptide-specific CTL precursors by IFN-γ release assay and tumor reactivity by 51Cr release assay. Delayed type hypersensitivity was also measured. For humoral response, pre and postvaccination (sixth) sera were provided for measurement of peptide-reactive IgG by an ELISA. Results: The median survival time and 1-year survival rate of the total cases were 346 ± 64.9 days and 44.6%, respectively, and those of patients vaccinated more than six times (n = 91) were 409 ± 15 days and 54.4%, respectively. In these 91 patients, the overall survival of patients whose sera showed increased levels of peptide-reactive IgG (n = 60) was significantly more prolonged (P = 0.0003) than that of patients whose sera did not (n = 31), whereas none of cellular responses correlated with overall survival. Conclusions: Peptide-specific IgG in postvaccination sera could be a suitable laboratory maker for the prediction of prolonged survival in advanced cancer patients vaccinated with peptides based on pre-existing CTL precursors.

Synthetic MicroRNA Designed to Target Glioma-Associated Antigen 1 Transcription Factor Inhibits Division and Induces Late Apoptosis in Pancreatic Tumor Cells

Purpose: To determine whether the synthetic microRNAs (miRNA) could effectively target tumor cells we designed several miRNA complementary to glioma-associated antigen-1 (Gli-1) mRNA and investigated their ability to inhibit tumor cell proliferation. The sonic hedgehog pathway is an early and late mediator of tumorigenesis in epithelial cancers. Activation of sonic hedgehog signaling seems to precede transformation of tissue stem cells to cancerous stem cells, with the Gli-1 transcription factor functioning as a mediator of environmental signals. Inhibiting cancer cell proliferation by targeting the Gli-1 effector pathway is difficult to achieve by chemotherapeutic agents or short interfering RNA. Experimental Design: We hypothesized that targeting the 3′-untranslated region of Gli-1 mRNA would effectively inhibit tumor cell proliferation. To test this hypothesis, we used synthetic miRNAs of our own design and corresponding duplex/small temporal RNAs by introducing three-nucleotide loops in the 3′-untranslated region Gli-1 sequence of high GU content. Results: We found that miRNA (Gli-1-miRNA-3548) and its corresponding duplex (Duplex-3548) significantly inhibited proliferation of Gli-1+ ovarian (SK-OV-3) and pancreatic (MiaPaCa-2) tumor cells. The miRNAs mediated delayed cell division and activation of late apoptosis in MiaPaCa-2 cells. This is the first demonstration of inhibition of pancreatic tumor cell division by designed miRNA. Conclusions: Gli-1 miRNAs should significantly add to the general understanding of the mechanisms of metastasis and contribute toward the design of better treatments for epithelial cancers.

Expression of activated signal transducer and activator of transcription-3 predicts poor prognosis in cervical squamous-cell carcinoma

Background:Stat3 is a member of the Janus-activated kinase/STAT signalling pathway. It normally resides in the cytoplasm and can be activated through phosphorylation. Activated Stat3 (p-Stat3) translocates to the nucleus to activate the transcription of several molecules involved in cell survival and proliferation. The constitutive activation of Stat3 has been shown in various types of malignancies, and its expression has been reported to indicate a poor prognosis. However, the correlation between the constitutive activation of Stat3 and the prognosis of cervical cancer patients has not been reported.Methods:The immunohistochemical analysis of p-Stat3 expression was performed on tissues from 125 cervical squamous-cell carcinoma patients who underwent extended hysterectomy and pelvic lymphadenectomy, and the association of p-Stat3 expression with several clinicopathological factors and survival was investigated.Results:Positive p-Stat3 expression was observed in 71 of 125 (56.8%) cases and was significantly correlated with lymph node metastasis, lymph vascular space invasion, and large tumour diameter (>4 cm) by Fishers exact test. Kaplan–Meier survival analysis showed that p-Stat3 expression was statistically indicative of a poor prognosis for overall survival (P=0.006) and disease-free survival (P=0.010) by log-rank test.Conclusion:These data showed that p-Stat3 expression in cervical cancer acts as a predictor of poor prognosis.

Vaccination with predesignated or evidence-based peptides for patients with recurrent gynecologic cancers.

Two different trials of peptide vaccination were conducted for patients with recurrent gynecologic cancers. In the first regimen, four HLA-A24+ patients (two with cervical cancer and two with ovarian cancer) were vaccinated with peptides that were predesignated before vaccination. Three patients exhibited with a grade 1 adverse effect, and no clinical response was observed in any patients. In the second regimen, six HLA-A24+ and four HLA-A2+ patients (five with cervical cancer, one with endometrial cancer, one with uterine sarcoma, and three with ovarian cancer) were vaccinated with peptides (maximum four) to which preexisting cytotoxic T lymphocyte precursors in the periphery were confirmed before vaccination. With this regimen, grade 1 adverse effects were observed in eight patients, a grade 2 adverse effect in one patient, and a grade 3 adverse effect (ie, rectal bleeding) in one patient. However, this regimen was able to enhance peptide-specific cytotoxic T lymphocytes in seven of ten patients, and three of five cervical cancer patients showed objective tumor regression. Analysis of immunoglobulin G -reactive to administered peptides suggested that the induction of peptide-specific immunoglobulin G was correlated with clinical responses. Overall, these results suggest that peptide vaccination of patients showing evidence of preexisting peptide-specific cytotoxic T lymphocyte precursors could be superior to vaccination with predesignated peptides, and that the evidence-based regimen is applicable for clinical trials in treatment of patients with recurrent gynecologic cancers.

Identification of SART3‐derived peptides capable of inducing HLA‐A2‐restricted and tumor‐specific CTLs in cancer patients with different HLA‐A2 subtypes

We recently identified the SART3 antigen encoding shared tumor epitopes recognized by HLA‐A2402‐restricted and tumor‐specific CTLs. Our study investigated whether the SART3 antigen encodes peptides recognized by the HLA‐A2‐restricted CTLs. The HLA‐A2‐restricted and tumor‐specific CTL line recognized COS‐7 cells co‐transfected with the SART3 gene and either HLA‐A0201, ‐A0206 or ‐A0207 cDNA but not those co‐transfected with the SART3 gene and HLA‐A2402 or ‐A2601 cDNA. The 2 SART3 peptides at positions 302 to 310 and 309 to 317 possessed the ability to induce HLA‐A2‐restricted and tumor‐specific CTLs from peripheral blood mononuclear cells of cancer patients with various histological types and different HLA‐A2 subtypes. Therefore, these 2 peptides could be useful for specific immunotherapy of a relatively large number of HLA‐A2+ cancer patients. Int. J. Cancer 88:633–639, 2000.

Cap43/NDRG1/Drg-1 is a molecular target for angiogenesis and a prognostic indicator in cervical adenocarcinoma.

Cap43 is a nickel- and calcium-inducible gene that plays important roles in the primary growth of malignant tumors, as well as in invasion and metastasis, most likely through its ability to induce cellular differentiation. This study investigated associations of Cap43 expression with angiogenesis and other clinicopathological factors in cervical adenocarcinoma. The clinical records of 100 women who underwent surgery for cervical adenocarcinoma were reviewed retrospectively. Microvessel density and the expression of Cap43 and VEGF in the surgical specimens were evaluated immunohistochemically. The Cap43 expression level was significantly associated with angiogenesis, tumor diameter, stromal invasion, lymphovascular space invasion, lymph node metastasis, and histopathological differentiation. Kaplan-Meier analysis showed a significant association between the Cap43 expression level and survival: high Cap43 expression was related to poor survival. Our results suggest that increased expression of Cap43 is associated with angiogenesis and may be a poor prognostic indicator in women with cervical adenocarcinoma.

Melanoma differentiation-associated gene-7 (mda-7 )/interleukin (IL)-24 induces anticancer immunity in a syngeneic murine model

Previous studies have shown that the human melanoma differentiation-associated gene-7 (mda-7)/interleukin-24 (IL-24) has tumor-suppressor activity in vitro and in vivo. Additionally, in vitro studies using human peripheral blood mononuclear cells indicate that mda-7/IL-24 has TH1 cytokine-like activity. However, the individual properties of mda-7/IL-24 have been previously examined separately. Thus, there is not a single study that has examined both, antitumor and proimmune properties of mda-7/IL-24. Furthermore, the tumor suppressive activity and the cytokine activity of mda-7/IL-24 have not been previously tested in an immunocompetent setting. We therefore in the present study evaluated the antitumor and immune properties of mda-7/IL-24 in a murine syngeneic tumor model. In vitro, adenovirus-mediated mda-7 gene (Ad-mda7) transfer to murine fibrosarcoma (UV2237m; MCA16) and normal (10T1/2) cells significantly inhibited growth (P=0.001) and induced apoptosis in tumor cells but not in normal cells. In vivo, intratumoral administration of Ad-mda7 resulted in significant inhibition of tumor growth (P<0.05), with a subset of mice showing complete tumor regression. We next evaluated the immune potentiation activity of Ad-mda7 in a cancer vaccine model. UV2237m cells transfected with Ad-mda7 and injected into syngeneic immunocompetent C3H mice were unable to grow; however, they did grow in immunocompromised nude mice. These tumor-free C3H mice, when challenged with parental tumor cells experienced no tumor growth, suggesting induction of systemic immunity. Moreover, splenocytes prepared from vaccinated C3H mice demonstrated higher proliferative activity and produced elevated levels of TH1 cytokines compared with those from control mice. An in vitro subset analysis of splenocytes from vaccinated mice demonstrated a significant increase in the CD3+CD8+ but not the CD3+CD4+ cell population (P=0.019). Thus Ad-mda7 treatment of syngeneic tumors induces tumor cell death and promotes immune activation, leading to anticancer immunity.

Prostate Tumor Cells Infected with a Recombinant Influenza Virus Expressing a Truncated NS1 Protein Activate Cytolytic CD8+ Cells To Recognize Noninfected Tumor Cells

ABSTRACT Many viral oncolytic approaches against cancer are based on the ability of specific viruses to replicate in tumors expressing components of the constitutively activated Ras/mitogen-activated protein kinase (MAPK) pathways and/or inhibited or dysregulated alpha/beta interferon (IFN-α/β) response pathways. A major issue when considering these approaches is their applicability to tumors that lack activated Ras. To identify the effector mechanisms activated by oncolytic viruses, we investigated inhibition of proliferation of the prostate cancer line LNCap by the recombinant TR-NS1 influenza A virus, a genetically attenuated influenza A/PR8/34 virus expressing a truncated nonstructural protein (NS1) of 126 amino acids. LNCap cells lack constitutively activated MAPK, extracellular signal-regulated kinase (ERK), and p38 and are resistant to death by IFN-α. Truncation of the NS1 protein of influenza viruses is known to result in viral attenuation due to a reduced ability of the NS1 to inhibit the IFN-α/β response. Infection with TR-NS1 virus rapidly activated ERK-1 more than ERK-2 in LNCap cells. Importantly, TR-NS1 virus infection transiently inhibited cell proliferation and induced apoptosis in LNCap cells. Addition of peripheral blood mononuclear cells (PBMC) and interleukin 12 (IL-12) to TR-NS1 virus-infected LNCap cells (TR-NS1-LNCap) resulted in faster elimination of TR-NS1-LNCap cells compared with LNCap cells. Moreover, TR-NS1-LNCap cells induced IFN-γ in PBMC. The levels of IFN-γ were amplified by IL-12. TR-NS1-LNCap cells also induced tumor-lytic cytotoxic T lymphocytes (CTL). These CTL lysed noninfected LNCap cells in a CD8-dependent manner. Activation of cellular immunity to tumor cells by viruses is an intriguing effector pathway, which should be especially significant for elimination of human tumors that lack activated Ras.

UDP-Gal: βGlcNAc β1, 3-galactosyltransferase, polypeptide 3 (GALT3) is a tumour antigen recognised by HLA-A2-restricted cytotoxic T lymphocytes from patients with brain tumour

Patient prognosis in the case of malignant brain tumours is generally poor, despite significant improvements in the early detection of the tumours, and thus the development of new treatment modalities is needed. One of the most prominent modalities is specific immunotherapy, for which the elucidation of antigenic molecules of malignant brain tumours recognized by T cells is essential. We report here a gene, UDP-Gal: βGlcNAc β1, 3-galactosyltransferase, polypeptide 3, encoding three epitope peptides recognised by tumor-reactive cytotoxic T lymphocytes in an HLA-A2-restricted manner. Two of the three peptides possessed an ability to induce HLA-A2-restricted and tumour-reactive cytotoxic T lymphocytes from peripheral blood mononuclear cells of patients with brain tumours. These peptides may be useful in the peptide-based specific immunotherapy for patients with malignant brain tumours.