Transfection of human hepatoma cell lines with cloned HBV DNA resulted in the secretion of large amounts of hepatitis B surface antigen (HBsAg) and core‐related antigens (HBc/HBeAg) if well‐differentiated cell lines were employed. Synthesis of both viral antigens was the highest in cell line HuH‐7 and continued for approximately 25 days. Particles resembling hepatitis B virions (Dane particles) by morphology, density and by the presence of the preS1 surface antigen were released from the transfected HuH‐7 cells into the culture medium. These particles produced in vitro were also indistinguishable from the naturally occurring hepatitis B virions in containing the virus‐associated DNA polymerase and mature HBV genomes. Restriction analysis of these DNA molecules was compatible with the nucleotide sequence of the transfecting HBV DNA sequence. Viral surface antigens and core proteins present in the culture medium were fractionated and characterized by immunoprecipitation and SDS‐‐PAGE after labeling with [35S]methionine. Antisera specific for X‐gene products identified in cell extracts two hitherto unknown HBV gene products. This system thus provides a new approach to open questions regarding HBV‐related gene function and HBV replication.

Previous reports indicate that human hepatocytes do not express class I and class II MHC antigens. Our analyses on 10 human hepatocellular carcinoma (HCC) cell lines by immunofluorescence tests and RIA, demonstrate that all the human HCC cell lines tested express class I MHC antigens and among them, three poorly differentiated human HCC cell lines also express class II MHC antigens. Results of immunoprecipitation and/or Western blotting experiments indicate similarity in the chemical nature of both the class I and class II MHC antigens expressed by the human HCC cell lines and by a human B lymphoblastoid cell line Raji. Furthermore, a new variant form of class I antigen was detected in some of these HCC cell lines. Immunohistochemical studies of HCC tissues using the peroxidase-antiperoxidase staining method indicated that class I and class II antigens were detectable in 7 out of 11 and 3 out of 11 HCC tissues from patients, respectively. The availability of MHC class I antigen-positive cultured HCC cell lines, including the poorly differentiated lines that also express MHC class II antigen, has provided us with interesting models to study the relationship between expression of MHC antigen and transformation and differentiation of human hepatocytes. These studies will also allow us some insight into the role of MHC class I and class II antigen in the immunosensitivity and immunogenecity of HCC cells to the host-immune response.

The metastatic tumor antigen 1 (MTA1) protein is associated with tumor invasiveness and poor prognosis in patients with hepatocellular carcinoma (HCC), particularly in those with hepatitis B virus (HBV)-related HCC. Chronically woodchuck hepatitis virus (WHV)-infected woodchuck is an ideal animal model for studying the pathogenesis of HBV-associated liver diseases, including HCC. To investigate the roles of MTA1 in HBV-associated hepatocarcinogenesis in the woodchuck model, we cloned the woodchuck MTA1 (wk-MTA1) complementary (c)DNA and characterized its molecular functions. The sequence and organization of the wk-MTA1 protein were highly conserved among different species. Similar to its expression in human HCC, wk-MTA1 was upregulated in woodchuck HCC, as determined at RNA and protein levels. Furthermore, an MTA1-spliced variant, wk-MTA1dE4, was overexpressed in woodchuck HCC, and it was attributed to approximately 50% of the total transcripts. The percentage of wk-MTA1dE4-overexpressed woodchuck HCCs was higher than that of the total wk-MTA1-overexpressed HCCs (77.8% vs 61.1%) and wk-MTA1dE4 may represent a more sensitive marker than the total wk-MTA1 in woodchuck HCC. We overexpressed or knocked down wk-MTA1 in a woodchuck HCC cell line and demonstrated that wk-MTA1 could interact with the WHV X protein (WHx) and play indispensable roles in WHx-mediated NF-κB activation and tumor cell migration- and invasion-promoting activities. In conclusion, our results support the hypothesis that woodchuck HCC recapitulates HBV-associated HCC with respect to the molecular characteristics of MTA1 and provides new clues for conducting mechanistic studies of MTA1 in HBV-associated hepatocarcinogenesis, including the possible clinical significance of wk-MTA1dE4.

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459 Background: Suratadenoturev (OBP-301) is an oncolytic adenovirus that harbors a promoter of human telomerase reverse transcriptase ( hTERT) and is genetically modified to selectively replicate within and then lyse cancer cells. The aim of this study was to assess the safety and optimal dosage for intratumoral (IT) injection of OBP-301 in patients with advanced hepatocellular carcinoma (HCC). Methods: An open-label, non-comparative, phase I dose-escalation trial was performed in 20 patients who had refractory advanced HCC. OBP-301 was administered to the primary tumor using ultrasound guidance. A single IT injection of 1010 virus particles (VP) was administered to patients in the initial cohort (Cohort-1), and the subsequent single-dose cohorts received 1011 VP (Cohort-2), 1012 VP (Cohort-3), and 3×1012 VP (Cohort-4). A multiple dose cohort (Cohort-5) received 2×1012 VP × 3 times every 2 weeks. Each of the single-dose cohorts had 3 patients and there was a single escalating dose of OBP-301 from 1010 to 3×1012 VP. The multiple-dose cohort had 8 patients, and 6 of them received multiple escalating doses of 2×1012 VP ×3 times every 2 weeks. Results: There were 18 males and 2 females, and the median age was 59.39 years (range: 48.4–65.9). Patients had good tolerance of the single dose and multiple-dose regimens, and the maximum tolerated dose (MTD) was more than 6×1012 VP/patient. There was no evidence of toxicity with increasing dose, but there was a greater frequency of treatment-emergent adverse events (TEAEs) in Cohorts 4–5 than in Cohorts 1–3. The most common TEAEs related to OBP-301 were influenza-like illness (30%), pyrexia (15%), and fatigue, decreased platelet count, abdominal distension, and anemia (10% each). The overall intrahepatic mRECIST response occurred in 7 patients (39%) with confirmed stable disease (SD) and 11 (61%) with progressive disease (PD). The best target response occurred in 14 patients (78%) and 4 (22%) of them had PD. There was evidence of OBP-301 replication-dependent dissemination in the blood. Cohorts 4–5 had about 50% greater levels of CD8+ T cells in peripheral blood after OBP-301 injection. Conclusions: Multiple IT injections of OBP-301 are well-tolerated in advanced HCC. Although antitumor activity of the study medication alone would not be demonstrated obviously, SD observed as best local response was higher than as overall response. Improved anti-tumor efficacy can be achieved with adjusting viral injection volume to the target sites as well as with adopting the combination therapy with another immunothrapeutics in further study (JRCT ID: jRCT2033200223). Clinical trial information: NCT02293850.