HB-EGF Is a Promising Therapeutic Target for Lung Cancer with Secondary Mutation of EGFRT790M.

Advanced lung cancer is one of the most lethal malignancies. Many anticancer agents have been developed for lung cancer with epidermal growth factor receptor (EGFR) mutations, but its prognosis remains extremely poor. The development of molecularly-targeted therapies is required for patients with lung cancer with secondary mutation of the EGFR gene. In this study, in order to assess the validity of heparin-binding EGF-like growth factor (HB-EGF) as a therapeutic target for lung cancer with EGFR mutation, we examined the antitumor effects of a specific inhibitor (cross-reacting material 197; CRM197) on lung cancer cells with EGFR mutation. HB-EGF was the most predominantly expressed EGFR ligand in lung cancer cells with EGFR mutation. CRM197 induced significant cell apoptosis and marked suppression of tumorigenicity in lung cancer cells with single or double mutation of EGFR. These results suggest that HB-EGF is a rational target for the treatment of lung cancer with EGFR mutation.

Antitumour Effects of Intravenous Administration of BK-UM, a Novel Inhibitor of HB-EGF, in Ovarian Cancer Therapy.

BACKGROUND: Patients with ovarian cancer with high levels of heparin-binding epidermal growth factor-like growth factor have a poor prognosis. Here we assessed the pharmacokinetics and tumour-inhibiting effects of cross-reacting material 197, produced commercially as BK-UM, and examined the efficacy and safety of its intravenous (i.v.) administration. MATERIALS AND METHODS: BK-UM was administered to rats, and its serum levels were measured. Ovarian cancer cell lines were either intraperitoneally (i.p.) or subcutaneously administered into mice, to establish a mouse model of ovarian cancer. BK-UM was then administered i.p. or i.v., and its tumour-inhibiting effects were examined. RESULTS: Higher maximum serum concentration (Cmax) values resulted from i.v. administration, whereas longer time to maximum serum (Tmax) values resulted from i.p. administration. In the peritoneal dissemination model, i.p. administration inhibited tumour growth and increased survival rate, whereas in the subcutaneous model, i.v. administration significantly inhibited tumour growth compared to i.p. administration. CONCLUSION: Administration of BK-UM by i.v. is both efficacious and safe.

BK-UM in patients with recurrent ovarian cancer or peritoneal cancer: a first-in-human phase-I study.

BACKGROUND: BK-UM (CRM197) is a mutant form of diphtheria toxin and a specific inhibitor of heparin-binding epidermal growth factor-like growth factor (HB-EGF). We assessed the safety, pharmacokinetics, recommended dose, and efficacy of BK-UM in patients with recurrent ovarian cancer (OC) or peritoneal cancer (PC), and measured HB-EGF levels in serum and abdominal fluid after BK-UM administration. METHODS: Eleven patients with advanced or recurrent OC or PC were enrolled and treated with BK-UM via the intraperitoneal route. The dose was escalated (1.0, 2.0, 3.3, and 5.0 mg/m2) using a 3 + 3 design. RESULTS: Eight of 11 patients completed treatment. No dose-limiting toxicity (DLT) was experienced at dose levels 1 (1.0 mg/m2) and 2 (2.0 mg/m2). Grade 3 transient hypotension as an adverse event (defined as a DLT in the present study) was observed in two of four patients at dose level 3 (3.3 mg/m2). Treatment with BK-UM was associated with decreases in HB-EGF levels in serum and abdominal fluid in seven of 11 patients and five of eight patients, respectively. Clinical outcomes included a partial response in one patient, stable disease in five patients, and progressive disease in five patients. CONCLUSIONS: BK-UM was well tolerated at doses of 1.0 and 2.0 mg/m2, with evidence for clinical efficacy in patients with recurrent OC or PC. A dose of 2.0 mg/m2 BK-UM is recommended for subsequent clinical trials. TRIAL REGISTRATION: This trial was prospectively performed as an investigator-initiated clinical trial. The trial numbers are UMIN000001002 and UMIN000001001, with registration dates of 1/30/2008 and 2/4/2008, respectively. UMIN000001001 was registered as a trial for the continuous administration of BK-UM after UMIN000001002 .

Anti-tumor Effect of Intravenous Administration of CRM197 for Triple-negative Breast Cancer Therapy.

BACKGROUND/AIM: Heparin-binding epidermal growth factor-like growth factor (HB-EGF), which belongs to the epidermal growth factor family, is a rational therapeutic target for triple-negative breast cancer (TNBC). This study aimed to assess the anti-tumor efficacy of intravenous (i.v.) HB-EGF-specific inhibitor (CRM197) for TNBC. MATERIALS AND METHODS: NOD/SCID mice were subcutaneously injected withTNBC cells, MDA-MB-231, and, then, treated with i.v. CRM197 in either dose- or frequency-dependent manners, using an advanced cancer model and an adjuvant therapy model. Tumor volume and mouse body weight were calculated weekly. Statistical significance was assessed by the Mann-Whitney U-test. RESULTS: Mice that received i.v. CRM197 showed a significant anti-tumor effect in dose- and frequency-dependent manners in both models. However, their body weight did not differ significantly among groups. CONCLUSION: These results suggest that i.v. CRM197 is an effective treatment for TNBC.

Evaluation of varicella zoster virus-specific cell-mediated immunity by using an interferon-γ enzyme-linked immunosorbent assay.

BACKGROUND: Administration of the varicella vaccine induces both varicella-zoster virus (VZV)-specific humoral and cell-mediated immunity (CMI). OBJECTIVE: To assess VZV-CMI, we developed an interferon γ enzyme-linked immunosorbent assay (IFN-γ ELISA) that measures the quantity of total IFN-γ in culture supernatants of human peripheral blood mononuclear cells. STUDY DESIGN: We evaluated this method by comparing the pre- and post-vaccination immune response in peripheral blood mononuclear cells of 30 healthy children who were administered an initial varicella vaccination at Konan Kosei hospital. RESULTS: IFN-γ ELISA showed well-validated results; CMI was not detectable pre-immunization but became detectable post-immunization. Seroconversion was detected in 92.6% of subjects by the immune adherence hemagglutination test; however, half of the subjects did not display an increase in CMI levels. We also compared the incidence of breakthrough varicella and herpes zoster development between CMI post-positive and post-negative vaccinees at 1-2years after the last VZV vaccination. Eight subjects had a history of varicella or herpes zoster exposure post-VZV vaccination. Two of them with post-negative CMI contracted breakthrough varicella 15-16months after the last vaccination, even though they had sufficient VZV-specific antibody levels to be considered seropositive and seroprotected. Conversely, the others with post-positive CMI did not contract breakthrough varicella, despite experiencing extensive VZV exposure through casual contact with playmates and family. CONCLUSIONS: The CMI data generated by this IFN-γ ELISA may accurately reflect real-world immune status, and CMI may be closely related to immunoprotection against breakthrough varicella development.

The efficacy of inactivated West Nile vaccine (WN-VAX) in mice and monkeys.

BACKGROUND: West Nile virus (WNV) belonging to the genus Flavivirus of the family Flaviviridae causes nervous system disorder in humans, horses and birds. Licensed WNV vaccines are available for use in horses but not for humans. We previously developed an inactivated West Nile virus vaccine (WN-VAX) using a seed virus from West Nile virus (WNV NY99) that was originally isolated in New York City in 1999. In this study, we report the immunogenicity of WN-VAX in both mice and non-human primates. FINDINGS: The WN-VAX immunized mice showed protection against lethal infection with WNV NY99. The challenge test performed on mice passively immunized with serum from other mice that were previously immunized with WN-VAX confirmed that the neutralizing antibody titers of more than 1log10 protected the passively immunized mice from WNV lethal infection. Furthermore, monkeys (Macaca fascicularis) immunized three times with 2.5 µg, 5 µg or 10 µg/dose of WN-VAX exhibited neutralizing antibodies in their sera with titers of more than 2log10 after the second immunization. CONCLUSIONS: The WN-VAX was protective in mice both by active and passive immunizations and was immunogenic in monkeys. These results suggest that the vaccine developed in this study may be a potential WNV vaccine candidate for human use.

Contribution of transcription factor, SP1, to the promotion of HB-EGF expression in defense mechanism against the treatment of irinotecan in ovarian clear cell carcinoma.

Ovarian clear cell carcinoma (OCCC) is a worst histological subtype than other ovarian malignant tumor. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a promising target for ovarian cancer therapy. The aims of this study were to validate the efficacy of HB-EGF-targeted therapy for OCCC and to identify the transcription factor that contributed to the induction of HB-EGF by SN38 treatment in OCCC cells. HB-EGF was highly expressed in OCCC cells, and an increase of HB-EGF was induced by SN38 which had only antitumor effect among conventional anticancer agents on OCCC. A specific inhibitor of HB-EGF, a cross-reacting material 197 (CRM197), led to a synergistic increase in the number of apoptotic OCCC cells with the treatment of SN38. The luciferase assay with 5'-deletion promoter constructs identified a GC-rich element between -125 and -178 (the distal transcription start site was denoted +1) as a cis-regulatory region, and the treatment of SN38 induced luciferase activity in this region. An in silico and chromatin immunoprecipitation analysis estimated that SP1 bound to the cis-regulatory region of HB-EGF in OCCC cells. Real-time PCR and cell viability assays showed that the transfection of a small interfering RNA targeting SP1 suppressed the expression of HB-EGF induced by SN38, resulting in the enhanced sensitivity of SN38. Taken together, these results indicate that induction of HB-EGF expression contributed to defense mechanism against treatment of SN38 through the transcriptional activity of SP1 in OCCC cells.

Pre-clinical study of BK-UM, a novel inhibitor of HB-EGF, for ovarian cancer therapy.

BACKGROUND/AIM: Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of the epidermal growth factor family, is a target for ovarian cancer therapy. The present study investigated the administration schedule of BK-UM, an anticancer agent targeting HB-EGF. MATERIALS AND METHODS: The ovarian cancer cell line, RMG-I, was injected subcutaneously into five-week-old female nude mice. The BK-UM was administered intraperitoneally, using three administration schedules with different doses. The tumor volume was calculated every week. Statistical significance was assessed using the Mann-Whitney U-test. RESULTS: At doses >0.1 mg/kg, BK-UM displayed significant antitumor effects, although the antitumor effects and body weights of mice did not significantly differ by dose or by three different administration schedules. At a dose <0.1 mg/kg, however, BK-UM had little inhibitory effect on tumor growth. CONCLUSION: Daily administration of BK-UM, which has a potentially dose-dependent antitumor effect, may be the optimal schedule for clinical application.

Superior immunogenicity of a freeze-dried, cell culture-derived Japanese encephalitis vaccine (inactivated).

Japanese encephalitis is an infectious disease caused by the Japanese encephalitis virus, which is widespread throughout Asia. The worldwide incidence is 50,000 cases per year. There is no specific treatment available, but inactivated mouse brain-derived vaccine was used from the 1950s to prevent infection. However, quality control of mouse brain-derived vaccines is difficult, and therefore a new freeze-dried, cell culture-derived Japanese encephalitis vaccine (inactivated) (JEBIK V; development code: BK-VJE) was developed. In this paper, we report an analysis of neutralizing antibody titers in vaccinated subjects enrolled in clinical study of BK-VJE at various doses, and study of BK-VJE with the mouse brain-derived vaccine as a control. The results show that BK-VJE has superior immunogenicity compared to mouse brain-derived vaccine.

Characterization of neutralizing antibodies in adults after intranasal vaccination with an inactivated influenza vaccine.

The levels and properties of neutralizing antibodies in nasal wash and serum collected from five healthy adults were examined after intranasal administration of an A/Uruguay/716/2007 (H3N2) split vaccine (45 µg hemagglutinin (HA) per dose; five doses, with an interval of 3 weeks between each dose). Prior to the assays, nasal wash samples were concentrated so that the total amount of antibodies was equivalent to about 1/10 of that found in the natural nasal mucus. Vaccination induced virus-specific neutralizing antibody responses, which increased with the number of vaccine doses given. Neutralizing antibodies were produced more efficiently in the nasal passages than in the serum: A ≥4-fold increase in nasal neutralization titres was observed after the second vaccination in four out of five subjects, whereas a rise in serum neutralization titres was observed only after the fifth vaccination. Nasal and serum neutralizing antibodies were mainly found in the polymeric IgA and monomeric IgG fractions, respectively, after gel filtration. Taken together, these results suggest that intranasal administration of an inactivated split vaccine induces high levels of nasal neutralizing antibodies (primarily polymeric IgA) and low levels of serum neutralizing antibodies (primarily monomeric IgG).

Antitumor effects of CRM197, a specific inhibitor of HB-EGF, in T-cell acute lymphoblastic leukemia.

The therapeutic outcome for T-cell acute lymphoblastic leukemia (T-ALL) remains poor; thus, novel, targeted therapies are urgently needed. Recently, we showed that heparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of the EGF family, is a promising target for the treatment of various types of cancer. The aim of the present study was to investigate whether HB-EGF is a therapeutic target for T-ALL, and to further elucidate the antitumor effects of a specific inhibitor of HB-EGF, cross-reacting material 197 (CRM197). We elucidated the expression of HB-EGF in T-ALL cell lines, and evaluated the effect of CRM197 on these cells alone or in combination with anticancer agent. The expression of EGFR and EGFR ligands was determined by flow cytometry, RT-PCR and real-time quantitative PCR. Induction of apoptosis was assessed by TUNEL assay. HB-EGF was strongly expressed by T-ALL cell lines, and the expression of both HB-EGF and EGFR was enhanced by doxorubicin. CRM197 induced apoptosis, and furthermore, the combination of CRM197 plus doxorubicin enhanced cytotoxicity in a T-ALL cell line. These results suggest that HB-EGF is a promising therapeutic target for T-ALL.

Development and evaluation of a formalin-inactivated West Nile Virus vaccine (WN-VAX) for a human vaccine candidate.

A formalin-inactivated West Nile Virus (WNV) vaccine (WN-VAX) derived from the WNV-NY99 strain was tested for its safety, efficacy, dilution limit for complete protection, and cross-neutralization. Safety tests performed with experimental animals, bacteria, or cultured cell lines showed no evidence of short- or long-term adverse effects. WN-VAX also protected 100% of 4-week-old mice against a lethal challenge from the WNV-NY99 strain after two doses of intraperitoneal inoculation-even when the vaccine was diluted to 3.2ng/dose. Moreover, very limited cross-neutralization activity against Japanese encephalitis virus, Dengue virus, Murray Valley encephalitis virus, Yellow fever virus or St. Louis encephalitis virus was observed. Therefore, the WN-VAX satisfies the requirements for human trials planned to be done in Japan.

Purification and characterization of a clostripain-like protease from a recombinant Clostridium perfringens culture.

Clostridium perfringens produces a homologue of clostripain (Clo), the arginine-specific endopeptidase of Clostridium histolyticum. To determine the biochemical and biological properties of the C. perfringens homologue (Clp), it was purified from the culture supernatant of a recombinant C. perfringens strain by cation-exchange chromatography and ultrafiltration. Analysis by SDS-PAGE, N-terminal amino acid sequencing and TOF mass spectrometry revealed that Clp consists of two polypeptides comprising heavy (38 kDa) and light (16 kDa or 15 kDa) chains, and that the two light chains differ in the N-terminal cleavage site. This difference in the light chain did not affect the enzymic activity toward N-benzoyl-l-arginine p-nitroanilide (Bz-l-arginine pNA), as demonstrated by assaying culture supernatants differing in the relative ratio of the two light chains. Although the purified Clp preferentially degraded Bz-dl-arginine pNA rather than Bz-dl-lysine pNA, it degraded the latter more efficiently than did Clo. Clp showed 2.3-fold higher caseinolytic activity than Clo, as expected from the difference in substrate specificity. Clp caused an increase in vascular permeability when injected intradermally into mice, implying a possible role of Clp in the pathogenesis of clostridial myonecrosis.

Application of the arming system for the expression of the 380R antigen from red sea bream iridovirus (RSIV) on the surface of yeast cells: a first step for the development of an oral vaccine.

The cell surface is a functional interface between the inside and the outside of the cell. Moreover, cells have systems for anchoring surface specific proteins and for confining surface proteins to particular domains on the cell surface. For use in bioindustrial processes applied to oral vaccination, we consider that cell-surface display systems must be useful and that the yeast Saccharomyces cerevisiae, the most suitable microorganism for practical purposes, is available as a host for genetic engineering because it can be subjected to many genetic manipulations. In particular, the rigid structure of the cell makes the yeast suitable for several of the applications. In this study, we describe the expression of one of the target antigens, 380R, from the red sea bream iridovirus (RSIV), which is one of the most common viral diseases in the cultured marine fish Pagrus major in Japan, using the arming yeast system and aiming at its application for oral vaccination. We first performed the molecular cloning and expression of the 380R antigen from RSIV in Escherichia coli. The nucleotide sequence of the 380R antigen was composed of an open reading frame (ORF) of 1360 bp encoding a protein of 453 residues. To prepare a specific antibody against the 380R antigen, the recombinant protein was overexpressed and purified in E. coli. As a result of indirect immunofluorescence with the specific antibody, we could observe the expression of the 380R antigen on the surface of the yeast cells. Thus, we have successfully prepared the source of an oral vaccine using cell-surface display technology in yeast.