Junctional adhesion molecule 3 is a potential therapeutic target for small cell lung carcinoma.

There are few effective therapies for small cell lung carcinoma (SCLC); thus, we need to develop novel and efficacious treatments. We hypothesized that an antibody-drug conjugate (ADC) could be a promising option for SCLC. Several publicly available databases were used to demonstrate the extent to which junctional adhesion molecule 3 (JAM3) mRNA was expressed in SCLC and lung adenocarcinoma cell lines and tissues. Three SCLC cell lines, Lu-135, SBC-5, and Lu-134 A, were selected and examined for JAM3 protein expression by flow cytometry. Finally, we examined the response of the three SCLC cell lines to a conjugate between an anti-JAM3 monoclonal antibody HSL156 (developed in-house) and the recombinant protein DT3C, which consists of diphtheria toxin lacking the receptor-binding domain but containing the C1, C2, and C3 domains of streptococcal protein G. In silico analyses revealed that JAM3 mRNA was expressed higher in SCLC cell lines and tissues than in those of lung adenocarcinoma. As expected, all the three SCLC cell lines examined were positive for JAM3 at the mRNA and protein levels. Consequently, control SCLC cells, but not JAM3-silenced ones, were highly sensitive to HSL156-DT3C conjugates, resulting in dose- and time-dependent decreased viability. Finally, silencing JAM3 alone suppressed the growth of all SCLC cell lines examined. Taken together, these findings suggest that an ADC targeting JAM3 could represent a new approach to treating SCLC patients.

Anthrax toxin receptor 2 is a potential therapeutic target for non-small cell lung carcinoma with MET exon 14 skipping mutations.

Although MET tyrosine kinase inhibitors (TKIs) are generally effective against non-small cell lung carcinoma (NSCLC) with MET exon 14 skipping mutations (METΔex14), resistance to MET TKIs can occur, indicating the need to develop other therapeutic options. We found that Hs-746 T cells, which harbor METΔex14 plus amplification, were able to survive and grow in the absence of MET signaling, exhibiting primary resistance to MET TKIs. We also found a moderately positive correlation between MET and anthrax toxin receptor 2 (ANTXR2) mRNA expression in NSCLC cell lines using data from the Cancer Dependency Map database. As expected, Hs-746 T cells were positive for ANTXR2 expression. We used an antibody-drug conjugate (ADC) analog in the form of an anti-ANTXR2 monoclonal antibody, H8R23, conjugated to DT3C recombinant protein which consists of diphtheria toxin (DT) lacking the receptor-binding domain but containing the C1, C2, and C3 domains of streptococcal protein G (3C). H8R23-DT3C conjugates, which function in vitro like an ADC, induced Hs-746 T cells to undergo apoptosis, resulting in decreased viability. These findings collectively suggest that an ADC targeting ANTXR2 could be effective for the treatment of METΔex14-positive NSCLC.

Antibody Screening System Using a Herpes Simplex Virus (HSV)-Based Probe To Identify a Novel Target for Receptor-Retargeted Oncolytic HSVs.

Herpes simplex virus (HSV) is a promising tool for developing oncolytic virotherapy. We recently reported a platform for receptor-retargeted oncolytic HSVs that incorporates single-chain antibodies (scFvs) into envelope glycoprotein D (gD) to mediate virus entry via tumor-associated antigens. Therefore, it would be useful to develop an efficient system that can screen antibodies that might mediate HSV entry when they are incorporated as scFvs into gD. We created an HSV-based screening probe by the genetic fusion of a gD mutant with ablated binding capability to the authentic HSV entry receptors and the antibody-binding C domain of streptococcal protein G. This engineered virus failed to enter cells through authentic receptors. In contrast, when this virus was conjugated with an antibody specific to an antigen on the cell membrane, it specifically entered cells expressing the cognate antigen. This virus was used as a probe to identify antibodies that mediate virus entry via recognition of certain molecules on the cell membrane other than authentic receptors. Using this method, we identified an antibody specific to epiregulin (EREG), which has been investigated mainly as a secreted growth factor and not necessarily for its precursor that is expressed in a transmembrane form. We constructed an scFv from the anti-EREG antibody for insertion into the retargeted HSV platform and found that the recombinant virus entered cells specifically through EREG expressed by the cells. This novel antibody-screening system may contribute to the discovery of unique and unexpected molecules that might be used for the entry of receptor-retargeted oncolytic HSVs.IMPORTANCE The tropism of the cellular entry of HSV is dependent on the binding of the envelope gD to one of its authentic receptors. This can be fully retargeted to other receptors by inserting scFvs into gD with appropriate modifications. In theory, upon binding to the engineered gD, receptors other than authentic receptors should induce a conformational change in the gD, which activates downstream mechanisms required for viral entry. However, prerequisite factors for receptors to be used as targets of a retargeted virus remain poorly understood, and it is difficult to predict which molecules might be suitable for our retargeted HSV construct. Our HSV-based probe will allow unbiased screening of antibody-antigen pairs that mediate virus entry and might be a useful tool to identify suitable pairs for our construct and to enhance our understanding of virus-cell interactions during infection by HSV and possibly other viruses.

[Cavernous Hemangioma Originating in the Left Atrial Appendage:Report of a Case].

A 66-year-old male with hypertension was referred for evaluation of abnormal find chest X-ray. A computed tomography (CT) scan revealed a solitary pericardial mass with a diameter of 5 cm, located in the left atrioventricular groove. It showed solid but unevenly enhanced contents suggesting a well vascularized tumor originating in either a part of the left heart or the pericardium. As magnetic resonance imaging showed a clear boundary between the tumor and the pericardium, cardiac origin was suspected. Surgical removal of the tumor was performed via median sternotomy. The tumor originated from the lateral aspect of the left atrial appendage, having a base of 10 mm in diameter. The tumor was fully excised with an associated left atrial cuff under cardiopulmonary bypass. The postoperative course was uneventful. The tumor was histopathologically diagnosed as cavernous hemangioma originating in the left atrial wall. There has been no sign of recurrence for four years following surgery.

Immunoregulatory influence of abundant MFG-E8 expression by esophageal cancer treated with chemotherapy.

Milk fat globule-epidermal growth factor factor 8 (MFG-E8) is secreted from macrophages and is known to induce immunological tolerance mediated by regulatory T cells. However, the roles of the MFG-E8 that is expressed by cancer cells have not yet been fully examined. Expression of MFG-E8 was examined using immunohistochemistry in surgical samples from 134 patients with esophageal squamous cell carcinoma. The relationships between MFG-E8 expression levels and clinicopathological factors, including tumor-infiltrating lymphocytes, were evaluated. High MFG-E8 expression was observed in 23.9% of the patients. The patients with tumors highly expressing MFG-E8 had a significantly higher percentage of neoadjuvant chemotherapy (NAC) history (P < .0001) and shorter relapse-free survival (P = 0.012) and overall survival (OS; P = .0047). On subgroup analysis, according to NAC history, patients with high MFG-E8 expression had significantly shorter relapse-free survival (P = .027) and OS (P = .0039) only when they had been treated with NAC. Furthermore, tumors with high MFG-E8 expression had a significantly lower ratio of CD8+ T cells/regulatory T cells in tumor-infiltrating lymphocytes (P = .042) only in the patients treated with NAC, and those with a lower ratio had a shorter OS (P = .026). High MFG-E8 expression was also found to be an independent prognostic factor in multivariate analysis. The abundant MFG-E8 expression in esophageal squamous cell carcinoma might have a negative influence on the long-term survival of patients after chemotherapy by affecting T-cell regulation in the tumor microenvironment.

Herpes simplex viral-vector design for efficient transduction of nonneuronal cells without cytotoxicity.

The design of highly defective herpes simplex virus (HSV) vectors for transgene expression in nonneuronal cells in the absence of toxic viral-gene activity has been elusive. Here, we report that elements of the latency locus protect a nonviral promoter against silencing in primary human cells in the absence of any viral-gene expression. We identified a CTCF motif cluster 5' to the latency promoter and a known long-term regulatory region as important elements for vigorous transgene expression from a vector that is functionally deleted for all five immediate-early genes and the 15-kb internal repeat region. We inserted a 16.5-kb expression cassette for full-length mouse dystrophin and report robust and durable expression in dystrophin-deficient muscle cells in vitro. Given the broad cell tropism of HSV, our design provides a nontoxic vector that can accommodate large transgene constructs for transduction of a wide variety of cells without vector integration, thereby filling an important void in the current arsenal of gene-therapy vectors.

Angiotensin-converting enzyme 2 is a potential therapeutic target for EGFR-mutant lung adenocarcinoma.

EGFR-mutant lung adenocarcinomas contain a subpopulation of cells that have undergone epithelial-to-mesenchymal transition and can grow independently of EGFR. To kill these cancer cells, we need a novel therapeutic approach other than EGFR inhibitors. If a molecule is specifically expressed on the cell surface of such EGFR-independent EGFR-mutant cancer cells, it can be a therapeutic target. We found that a mesenchymal EGFR-independent subline derived from HCC827 cells, an EGFR-mutant lung adenocarcinoma cell line, expressed angiotensin-converting enzyme 2 (ACE2) to a greater extent than its parental cells. ACE2 was also expressed at least partially in most of the primary EGFR-mutant lung adenocarcinomas examined, and the ACE2 expression level in the cancer cells was much higher than that in normal lung epithelial cells. In addition, we developed an anti-ACE2 mouse monoclonal antibody (mAb), termed H8R64, that was internalized by ACE2-expressing cells. If an antibody-drug conjugate consisting of a humanized mAb based on H8R64 and a potent anticancer drug were produced, it could be effective for the treatment of EGFR-mutant lung adenocarcinomas.

Syncytial Mutations Do Not Impair the Specificity of Entry and Spread of a Glycoprotein D Receptor-Retargeted Herpes Simplex Virus.

Membrane fusion, which is the key process for both initial cell entry and subsequent lateral spread of herpes simplex virus (HSV), requires the four envelope glycoproteins gB, gD, gH, and gL. Syncytial mutations, predominantly mapped to the gB and gK genes, confer hyperfusogenicity on HSV and cause multinucleated giant cells, termed syncytia. Here we asked whether interaction of gD with a cognate entry receptor remains indispensable for initiating membrane fusion of syncytial strains. To address this question, we took advantage of mutant viruses whose viral entry into cells relies on the uniquely specific interaction of an engineered gD with epidermal growth factor receptor (EGFR). We introduced selected syncytial mutations into gB and/or gK of the EGFR-retargeted HSV and found that these mutations, especially when combined, enabled formation of extensive syncytia by human cancer cell lines that express the target receptor; these syncytia were substantially larger than the plaques formed by the parental retargeted HSV strain. We assessed the EGFR dependence of entry and spread separately by using direct entry and infectious center assays, respectively, and we found that the syncytial mutations did not override the receptor specificity of the retargeted viruses at either stage. We discuss the implications of these results for the development of more effective targeted oncolytic HSV vectors. IMPORTANCE: Herpes simplex virus (HSV) is investigated not only as a human pathogen but also as a promising agent for oncolytic virotherapy. We previously showed that both the initial entry and subsequent lateral spread of HSV can be retargeted to cells expressing tumor-associated antigens by single-chain antibodies fused to a receptor-binding-deficient envelope glycoprotein D (gD). Here we introduced syncytial mutations into the gB and/or gK gene of gD-retargeted HSVs to determine whether viral tropism remained dependent on the interaction of gD with the target receptor. Entry and spread profiles of the recombinant viruses indicated that gD retargeting does not abolish the hyperfusogenic activity of syncytial mutations and that these mutations do not eliminate the dependence of HSV entry and spread on a specific gD-receptor interaction. These observations suggest that syncytial mutations may be valuable for increasing the tumor-specific spreading of retargeted oncolytic HSV vectors.

[Concomitant Operations for Thoracic Aortic Aneurysm and Myasthenia Gravis;Report of a Case].

A 77-year-old man, who had been under medical treatment for myasthenia gravis without thymoma, was diagnosed with aortic arch aneurysm. He underwent total aortic arch replacement and total resection of the thymus through median sternotomy. His symptoms relating to myasthenia gravis dramatically disappeared after the surgery. The serum anti-acetyl chorine receptor antibody decreased from 2.7 to 0.7 nmol/l (N<0.2) with the reduction of oral predonisolone from 12.5 to 5 mg/day at 4 years after the surgery. The concomitant operations significantly improved his quality of life.

Use of miRNA response sequences to block off-target replication and increase the safety of an unattenuated, glioblastoma-targeted oncolytic HSV.

Glioblastoma multiforme (GBM) is an aggressive brain cancer for which there is no effective treatment. Oncolytic HSV vectors (oHSVs) are attenuated lytic viruses that have shown promise in the treatment of human GBM models in animals, but their efficacy in early phase patient trials has been limited. Instead of attenuating the virus with mutations in virulence genes, we engineered four copies of the recognition sequence for miR-124 into the 3'UTR of the essential ICP4 gene to protect healthy tissue against lytic virus replication; miR-124 is expressed in neurons but not in glioblastoma cells. Following intracranial inoculation into nude mice, the miR-124-sensitive vector failed to replicate or show overt signs of pathogenesis. To address the concern that this safety feature may reduce oncolytic activity, we inserted the miR-124 response elements into an unattenuated, human receptor (EGFR/EGFRvIII)-specific HSV vector. We found that miR-124 sensitivity did not cause a loss of treatment efficiency in an orthotopic model of primary human GBM in nude mice. These results demonstrate that engineered miR-124 responsiveness can eliminate off-target replication by unattenuated oHSV without compromising oncolytic activity, thereby providing increased safety.

[Pulmonary Artery Catheter Injured by Ablation Device during Maze Procedure].

We report a case of pulmonary artery catheter (PAC) injury by radio frequency device for maze procedure. A 64-year-old female with severe mitral insufficiency, tricuspid insufficiency and paroxysmal atrial fibrillation was scheduled for mitral valve repair, tricuspid annulo- plasty and maze procedure including right-sided maze. Under general anesthesia, a PAC was inserted to pul- monary artery (PA) uneventfully. After radio frequency maze procedure and mitral valve repair, PAC was removed from right atrium by the surgeon for tricus- pid annuloplasty. Thereafter, the surgeon reinserted the PAC under transesophageal echocardiographic guidance since PAC balloon could not be inflated. PA pressure and cardiac output were not shown despite other parameters were correct We removed the PAC and reinserted a new one after the surgery. The PAC was compressed at about 25 cm from the tip and it appears to have been injured during right-sided maze procedure with radio frequency device. Complications of PAC are well known, including PA rupture and suture entrapment to the right atrium. To best of our knowledge, this is the first reported case of PAC injury by radio frequency device. Fortunately the PAC was not torn in our case ; however, there might have been a risk of infection through the thermodilu- tion cable.

Development of a sensitive screening method for selecting monoclonal antibodies to be internalized by cells.

Antibody-drug conjugates (ADCs), drugs developed by conjugation of an anticancer agent to a monoclonal antibody (mAb), have lately attracted attention in cancer therapy because ADCs can directly bind cancer cells and kill them. Although mAbs for ADCs must be internalized by the target cells, few methods are available for screening mAbs for their ability to be internalized by cells. We have developed a recombinant protein, termed DT3C, which consists of diphtheria toxin (DT) lacking the receptor-binding domain but containing the C1, C2, and C3 domains of Streptococcus protein G (3C). When a mAb-DT3C conjugate, which functions in vitro like an ADC, reduces the viability of cancer cells, the mAb being tested must have been internalized by the target cells. DT3C can thus be a tool to identify efficiently and easily mAbs that can be internalized by cells, thereby enhancing the development of promising ADCs.

Novel mutations in gB and gH circumvent the requirement for known gD Receptors in herpes simplex virus 1 entry and cell-to-cell spread.

Both entry and cell-to-cell spread of herpes simplex virus (HSV) involve a cascade of cooperative interactions among the essential glycoproteins D, B, and H/L (gD, gB, and gH/gL, respectively) initiated by the binding of gD to a cognate HSV entry receptor. We previously reported that a variant (D285N/A549T) of glycoprotein B (gB:NT) enabled primary virus entry into cells that were devoid of typical HSV entry receptors. Here, we compared the activities of the gB:NT variant with those of a newly selected variant of glycoprotein H (gH:KV) and a frequently coselected gB variant (gB:S668N). In combination, gH:KV and gB:S668N enabled primary virus entry into cells that lacked established HSV entry receptors as efficiently as did gB:NT, but separately, each variant enabled only limited entry. Remarkably, gH:KV uniquely facilitated secondary virus spread between cells that lacked canonical entry receptors. Transient expression of the four essential entry glycoproteins revealed that gH:KV, but not gB:NT, induced fusion between cells lacking the standard receptors. Because the involvement of gD remained essential for virus spread and cell fusion, we propose that gH:KV mimics a transition state of gH that responds efficiently to weak signals from gD to reach the active state. Computational modeling of the structures of wild-type gH and gH:KV revealed relatively subtle differences that may have accounted for our experimental findings. Our study shows that (i) the dependence of HSV-1 entry and spread on specific gD receptors can be reduced by sequence changes in the downstream effectors gB and gH, and (ii) the relative roles of gB and gH are different in entry and spread.

Development of a novel efficient method to construct an adenovirus library displaying random peptides on the fiber knob.

Redirection of adenovirus vectors by engineering the capsid-coding region has shown limited success because proper targeting ligands are generally unknown. To overcome this limitation, we constructed an adenovirus library displaying random peptides on the fiber knob, and its screening led to successful selections of several particular targeted vectors. In the previous library construction method, the full length of an adenoviral genome was generated by a Cre-lox mediated in vitro recombination between a fiber-modified plasmid library and the enzyme-digested adenoviral DNA/terminal protein complex (DNA-TPC) before transfection to the producer cells. In this system, the procedures were complicated and time-consuming, and approximately 30% of the vectors in the library were defective with no displaying peptide. These may hinder further extensive exploration of cancer-targeting vectors. To resolve these problems, in this study, we developed a novel method with the transfection of a fiber-modified plasmid library and a fiberless adenoviral DNA-TPC in Cre-expressing 293 cells. The use of in-cell Cre recombination and fiberless adenovirus greatly simplified the library-making steps. The fiberless adenovirus was useful in suppressing the expansion of unnecessary adenovirus vectors. In addition, the complexity of the library was more than a 10(4) level in one well in a 6-well dish, which was 10-fold higher than that of the original method. The results demonstrated that this novel method is useful in producing a high quality live adenovirus library, which could facilitate the development of targeted adenovirus vectors for a variety of applications in medicine.

Effective treatment of an orthotopic xenograft model of human glioblastoma using an EGFR-retargeted oncolytic herpes simplex virus.

Glioblastoma multiforme (GBM) remains an untreatable human brain malignancy. Despite promising preclinical studies using oncolytic herpes simplex virus (oHSV) vectors, efficacy in patients has been limited by inefficient virus replication in tumor cells. This disappointing outcome can be attributed in part to attenuating mutations engineered into these viruses to prevent replication in normal cells. Alternatively, retargeting of fully replication-competent HSV to tumor-associated receptors has the potential to achieve tumor specificity without impairment of oncolytic activity. Here, we report the establishment of an HSV retargeting system that relies on the combination of two engineered viral glycoproteins, gD and gB, to mediate highly efficient HSV infection exclusively through recognition of the abundantly expressed epidermal growth factor receptor (EGFR) on glioblastoma cells. We demonstrate efficacy in vitro and in a heterotopic tumor model in mice. Evidence for systemically administered virus homing to the tumor mass is presented. Treatment of orthotopic primary human GBM xenografts demonstrated prolonged survival with up to 73% of animals showing a complete response as confirmed by magnetic resonance imaging. Our study describes an approach to HSV retargeting that is effective in a glioma model and may be applicable to the treatment of a broad range of tumor types.

[A new method for reversed bevel technique using single-branched graft in hemi-arch replacement].

We describe a reversed bevel technique in hemi-arch replacement with a single-branched graft that enables long elliptical distal anastomosis and easier proximal anastomosis under antegrade systemic perfusion. If the distance between the clamped graft and the proximal aorta is too short, it becomes challenging to perform the anastomosis by everting the end of the graft. Because we clamp the graft at the most distal site, the side branch ends up being located at the beveled graft site.This method ensures sufficient surgical view during proximal anastomosis.

Induction of necroptosis in multinucleated giant cells induced by conditionally replicating syncytial oHSV in co-cultures of cancer cells and non-cancerous cells.

Viral modifications enabling syncytium formation in infected cells can augment lysis by oncolytic herpes simplex viruses (oHSVs) which selectively kill cancer cells. In the case of receptor-retargeted oHSVs (RR-oHSVs) that exclusively enter and spread to cancer cells, anti-tumor effects can be enhanced in a magnitude of >100,000-fold by modifying the virus to a syncytial type (RRsyn-oHSV). However, when syncytia containing non-cancerous cells are induced by conditionally replicating syncytial oHSV (CRsyn-oHSV), syncytial death occurs at an early stage. This results in limited anti-tumor effects of the CRsyn-oHSV. Here, we investigated whether necroptosis is involved in death of the syncytia formed by the fusion of cancer cells and non-cancerous cells. Mixed-lineage kinase domain-like (MLKL), a molecule executing necroptosis, was expressed in all murine cancer cell lines examined, while receptor-interacting protein kinase 3 (RIPK3), which phosphorylates MLKL, was absent from most cell lines. In contrast, RIPK3 was expressed in non-cancerous murine fibroblast cell lines. When a CRsyn-oHSV-infected RIPK3-deficient cancer cell line was co-cultured with the fibroblast cell line, but not with the cancer cells themselves, MLKL was phosphorylated and syncytial death was induced. These results indicate that early necroptosis is induced in multinucleated giant cells formed by CRsyn-oHSV when they also contain non-cancerous cells.

The left ventricular "lining" technique for repair of ischemic ventricular septal rupture.

We describe a technique to repair ischemic ventricular septal rupture via a left ventriculotomy. It employs a large endoventricular patch as a "lining" over the locally patched septal defect and the free wall defect which is going to be roofed with an external patch. Both defects are then closed in double layers, holding a single continuous patch. The technique enhances the advantage of the left ventriculotomy in the repair and minimizes ventriculotomy-related morbidity.

Isolated left hepatic vein draining into the coronary sinus: a rare anomaly encountered during mitral valve surgery.

A left hepatic vein draining into the coronary sinus is an extremely rare congenital abnormality as a solitary cardiovascular malformation. We encountered the anomaly during the mitral valve surgery in a 72-year-old woman. The operation was successfully carried out using an additional suction directly in the left hepatic vein.

Bispecific adapter-mediated retargeting of a receptor-restricted HSV-1 vector to CEA-bearing tumor cells.

The safety and efficacy of viral therapies for solid tumors can be enhanced by redirecting the virus infection to tumor-specific cell-surface markers. Successful retargeting of herpes simplex virus type 1 (HSV-1) has been achieved using vectors that carry a modified envelope glycoprotein D (gD) engineered to interact directly with novel receptors. In addition, soluble bridging molecules (adapters) have been used to link gD indirectly to cell-specific receptors. Here, we describe the development of an adapter connecting gD to the common tumor antigen carcinoembryonic antigen (CEA). The adapter consisted of a CEA-specific single-chain antibody fused to the gD-binding region of the gD receptor, herpes virus entry mediator (HVEM). We used this adapter in combination with a vector that is detargeted for recognition of the widely expressed gD receptor nectin-1, but retains an intact binding region for the less common HVEM. We show that the adapter enabled infection of HSV-resistant Chinese hamster ovary (CHO) cells expressing ectopic CEA and nectin-1/CEA-bearing human gastric carcinoma cells that are resistant to the vector alone. We observed cell-to-cell spread following adapter-mediated infection in vitro and reduced tumor growth in vivo, indicating that this method of vector retargeting may provide a novel strategy for tumor-specific delivery of tumoricidal HSV.