Phase I clinical study of a peptide vaccination for hepatitis C virus-infected patients with different human leukocyte antigen-class I-A alleles.

Hepatitis C virus (HCV) infection has a high risk of liver cirrhosis and hepatocellular carcinoma at later stages. We recently identified a peptide derived from the HCV core protein capable of inducing both cellular and humoral responses to nearly all HCV-positive patients in Japan with different human leukocyte antigen (HLA)-class I-A alleles. To assess the safety and immune responses to this novel peptide, we conducted a phase I dose-escalation study of the vaccination for 26 HCV-positive patients who were either non-responders to the interferon-based therapy (n = 23) or refused it (n = 3). The regimen was well tolerated, with no severe vaccine-related toxicity. Twenty-five and 22 patients completed the first and second cycle vaccination (6 and 12 vaccine injections), respectively. After a series of six vaccine injections, peptide-specific CTL activity was augmented in peripheral blood mononuclear cells from 15 of 25 patient samples, with an expected optimal dose of 1 mg peptide. After 12 vaccine injections, peptide-specific IgG production was augmented in plasma from the majority of patients (15 of 22 patients) tested, but not in a dose-dependent fashion. There were two HCV RNA responders with >1 log declines. Among patients whose pre-vaccination levels of alanine aminotransferase and alpha feto-protein exceeded the normal ranges, a <30% decrease was found in 7 of 24 and three of six patients, respectively. Because of its tolerability and higher rate of immune boosting, this protocol is recommended for a phase II study to investigate its clinical efficacy.

Combination therapy of personalized peptide vaccination and low-dose estramustine phosphate for metastatic hormone refractory prostate cancer patients: an analysis of prognostic factors in the treatment.

The aim of this study was to investigate prognostic factors of patients with metastatic hormone refractory prostate cancer (HRPC) under combined administration of personalized peptide vaccination and low-dose estramustine phosphate (EMP). From February 2001 to July 2004, 58 men with metastatic HRPC received the combination therapy of personalized peptide vaccination and low-dose EMP. Conducted immune monitorings for those patients were peptide-specific cytotoxic T lymphocyte (CTL) precursor analysis by interferon-gamma production and peptide-reactive immunoglobulin G (IgG) by an enzyme-linked immunosorbent assay. Clinical responses and survival times were also evaluated. The combination therapy was well tolerated with no major adverse effects. Increased levels of CTL precursors and IgG responses to the vaccinated peptides were observed in 29 of 37 (78%) patients and in 36 of 41 (88%) patients tested, respectively. A prostate-specific antigen decline of at least 50% occurred in 24% of patients. The median survival time was 17 months (95% confidence interval, 12-25 months). Cox proportional hazards analysis showed that a low number of lymphocytes (p = 0.0075, odds ratio 2.700), a negative immunological activity response after the vaccination (p = 0.0185, odds ratio 2.658), and poor performance status (p = 0.0347, odds ratio 2.569) were independent predictors of disease death. These encouraging results show the need for further evaluation of the combination of personalized peptide vaccination and low dose of EMP for metastatic HRPC patients.

[A phase I study of combination-therapy using personalized peptide vaccine and UFT/UZEL for advanced or recurrent colorectal cancer].

We herein administered combination-therapy using a personalized vaccination and the oral administration of UFT/UZEL for the treatment of either advanced or recurrent colorectal cancer. Safety and the efficacy of this new treatment modality were evaluated. Eight patients were enrolled in this clinical trial from July 2005 to May 2006. Up to 4 kinds of peptide vaccines which were selected according to the immune response of each patient were injected hypodermically every week. At the same time, UFT (300 mg/m2/day) and UZEL (75 mg/day) were also administered orally for 28 days followed by a 7-day rest period. Regarding the adverse events, callosity, a type of leukopenia, and liver-dysfunction, such as hyperbilirubinemia, an increase in the aminotransferase were observed. During the clinical evaluation after the end of the 2 courses, 3 cases showed progressive disease (PD) while 5 cases showed stable disease (SD). Our findings indicate that additional peptide vaccine therapy with UFT/UZEL, can be administered without compromising the patient's safety. Moreover, the use of UFT/UZEL does not normally interfere with the normal course of immune-therapy.

Role for the double-stranded RNA activated protein kinase PKR in E2F-1-induced apoptosis.

The transcription factor E2F-1 induces cell cycle progression at the G1/S checkpoint, and deregulation of E2F-1 provokes apoptosis in a wide variety of malignant cells. To date only p14(ARF) and p73, a p53 homologue, have been identified as E2F-1-inducible genes capable of mediating an apoptotic response. Here we show that adenovirus-mediated E2F-1 overexpression in cancer cells induces expression and autophosphorylation of the double-stranded RNA-dependent protein kinase PKR leading to phosphorylation of its downstream target, the alpha-subunit of the eukaryotic translation initiation factor 2 (eIF-2alpha) and to apoptotic cell death. This PKR-dependent apoptosis occurs in cell lines with mutated p53 and in cell lines with mutated p53 and p73, and is significantly reduced by the chemical inhibition of PKR activation. Further, PKR(-/-) mouse embryo fibroblasts, but not PKR(+/+) mouse embryo fibroblasts, demonstrate significant resistance to E2F-1-induced apoptosis. We conclude that an important pathway of E2F-1-mediated apoptosis is dependent on PKR activation and does not require p53 or p73.

Characteristics of severe adverse events after peptide vaccination for advanced cancer patients: Analysis of 500 cases.

The purpose of this study was to investigate severe adverse events (SAEs) after therapeutic peptide vaccination for advanced cancer patients. We investigated SAEs following personalized peptide vaccinations in 500 advanced cancer patients, including 174 prostate, 74 colon, 51 pancreatic and 43 gastric cancer patients. The number of vaccination cycles varied widely, from 3 to 112. The severity of adverse events was scored according to the National Cancer Institute Common Terminology Criteria for Adverse Events version 3, and events with a grade of >3 were defined as SAEs and were evaluated by the Institutional Safety Evaluation Committee. A total of 215 SAEs in 102 patients were recorded during the vaccine trials. The main causes for these events were cancer progression (152 SAEs in 78 patients), combined cancer treatments other than vaccination (35 in 21 patients), diseases other than cancer (20 in 19 patients), peptide vaccines (6 in 6 patients) and suicide (1 in 1 patient). The 6 vaccine-related SAEs, all grade 3, consisted of skin reactions at each injection site, cellulitis around the injection site, edemas of the head and neck regions, colitis, rectal bleeding and bladder-vaginal fistulae. Both cellular and humoral responses to the vaccinated peptides were highly boosted in all 6 of these patients, indicating the involvement of augmented immune responses in these SAEs. The clinical responses in these 6 patients consisted of 2 partial responses and 4 stable diseases. The majority of SAEs after peptide vaccination for advanced cancer patients were caused by cancer progression. The appearance of vaccine-related SAEs, except inflammatory injection site reactions, was unexpected, and fortunately the incidence was very low. Our results suggest that physicians should be on guard for these rare SAEs associated with augmented immune responses.

Phase I clinical study of a personalized peptide vaccination for patients infected with hepatitis C virus (HCV) 1b who failed to respond to interferon-based therapy.

To assess the safety and immune responses to a personalized peptide vaccination of hepatitis C virus (HCV) 1b-derived peptides, 12 HCV1b-positive patients, who were unresponsive to interferon-based therapy, were enrolled in this study. The reactivity of the pre-vaccination peripheral blood T cells and plasma IgG to four vaccine candidate peptides capable of inducing cytotoxic T lymphocytes (CTLs) in HLA-A24(+) patients was examined and only the reactive peptides were then administered bi-weekly at three different dose settings. The study was well tolerated with no severe toxicity. Augmentation of peptide-specific CTL activity and IgG in response to at least one of the vaccinated peptides was observed after the 7th vaccination. Decrease of serum alanine aminotransferase and HCV-RNA levels after the 14th vaccination was also observed in five and three patients, respectively.

The mitochondrial apoptosis-inducing factor plays a role in E2F-1-induced apoptosis in human colon cancer cells.

BACKGROUND: Overexpression of the transcription factor E2F-1 provokes apoptosis in cancer cells; the mechanism, however, is not completely understood. We sought to evaluate E2F-1 gene therapy in human colon cancer and to investigate the apoptotic pathway involved. METHODS: Adenoviral vectors were used to transfect the E2F-1 gene (Ad5E2F-1) or the control gene luciferase (Ad5Luc) into four human colon carcinoma cell lines. Apoptosis was confirmed by flow cytometry and poly (ADP-ribose) polymerase cleavage. Expression of apoptotic factors was determined with Western blot analysis. Inhibitory assays were used to determine the involvement of caspases in the apoptotic pathway. RESULTS: Overexpression of E2F-1 was evident in all cells treated with Ad5E2F-1; upregulation of Bcl-2, and activation of caspases were noted. The apoptosis-inducing factor in the cytosolic fraction was markedly upregulated after Ad5E2F-1 treatment. E2F-1 overexpression inhibited proliferation and induced significant apoptosis in all cell lines (P <.005). This apoptotic response could be only partially blocked by caspase inhibitors. CONCLUSIONS: These findings demonstrate that E2F-1 induces apoptosis and inhibits proliferation in human colon cancer cell lines. The marked upregulation of apoptosis-inducing factor and the fact that E2F-1-induced apoptosis is incompletely blocked by caspase inhibitors suggest a caspase-independent pathway of E2F-1-mediated apoptosis, reported here for the first time.