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Background: Gastric cancer (GC) is the second most common cancer in Japan in terms of incidence and the third most common in terms of deaths. The onset and progression of cancer are generally suppressed by the action of immune cells, which is an inflammatory response. MDSCs are thought to promote cancer progression and invasion by suppressing the function of immune cells, making them potential therapeutic and diagnostic targets. We measured MDSCs in GC patients and observed more monocyte-derived MDSCs in peripheral blood in advanced GC than in early GC. However, granulocyte-derived MDSCs showed no difference in GC progression when markers for classical MDSCs were used. We considered that granulocyte-derived MDSCs were much more abundant than monocyte-derived MDSCs in peripheral blood and were involved in cancer progression. We hypothesized that we had a new granulocyte-derived marker for advanced GC that did not show differences in classical markers, but may have different functions, and aimed to identify a novel subset that may be a therapeutic and diagnostic target. Method: For GC patients, we used heparin blood samples provided by patients who consented to Juntendo University. We analyzed the expression of various cell surface proteins against populations of CD33high, CD66bhigh, and HLA-DRlow as classical markers of MDSCs derived from granulocytes of GC patients using FACS (Melody). MDSCs were isolated by a cell sorter and used for experiments such as RNA-Seq. Novel markers and involved cytokines were quantified in plasma components by cytometric bead array. Result: The chemokine receptors CCR2, CCR5, CXCR4, and CXCR5 were highly expressed in MDSCs of advanced GC patients. The ability to suppress T-cell activation was also found to be stronger in MDSCs with advanced GC. In addition, IL-10 produced by MDSCs was also more abundant in the plasma of patients with advanced GC, as well as chemokines according to chemokine receptors. Conclusion: The high expression of chemokine receptors on MDSCs in advanced GC may indicate that chemokines produced at the cancer site facilitate the migration of MDSCs. These novel subsets of MDSCs found in advanced GC suggest that they may be new therapeutic targets. Citation Format: Suguru Yamauchi, Takumi Iwasawa, Tomoaki Ito, Malcom V. Brock, Tetsu Fukunaga, Hajime Orita, Kazunori Kato. Analysis of MDSCs as a diagnostic and therapeutic target for gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5958.

The development and progression of cancers are frequently promoted by immunosuppressive cells, such as Treg and myeloid-derived suppressor cells (MDSCs) mediated by various immune checkpoint molecules. Recently, the immune checkpoint inhibitor anti-PD-1 neutralizing antibody has become available for the first-line treatment of gastric cancer (GC) in Japan. However, there is no correlation between PD-1 or PD-L1 expression in Treg or GC tissues and clinical responses to anti-PD-1 antibody. MDSCs are immature myeloid cells thought to suppress immune cell action, but the functional cell surface markers that define MDSCs correlated with cancer progression has not yet been clarified well. In this study, we identified a new subset of potential therapeutic and diagnostic targets for MDSCs in GC patients. Significantly higher levels of immune checkpoint molecules, PD-1 and PD-L1, were expressed on granulocytic MDSCs from advanced than early-stage of GC patients. Interestingly, we found that a subset of G-MDSCs expressed not only PD-1 but also PD-L1 and the pretreatment of G-MDSCs with anti-PD-1 antibody ameliorated T cell responses in vitro. We also found that G-MDSCs isolated from patients treated with anti-PD-1 antibody had a lower ability to suppress T cells. These data indicate that a novel subset of G-MDSCs expressing PD-1 found in advanced GC could be novel therapeutic targets. Citation Format: Takumi Iwasawa, Suguru Yamauchi, Tetsu Fukunaga, Hajime Orita, Kazunori Kato. Novel subset of granulocytic MDSCs as immunosuppressive regulators and therapeutic targets in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2889.

Objectives Galectin-3 is involved in various biological activities, including immune activations and fibrosis. Idiopathic inflammatory myopathies (IIMs) are autoimmune diseases of unknown aetiology, often complicated by interstitial lung disease (ILD). The aim of this study was to evaluate the expression of galectin-3 in sera and tissues of patients with IIM and assess the associations of galectin-3 with patient characteristics and disease activity. Results Serum galectin-3 levels were significantly higher in IIM patients than in healthy controls. The serum galectin-3 levels positively correlated with serum levels of inflammatory markers and proinflammatory cytokines/chemokines and the Myositis Intention-to-Treat Activity Index. Stratification analysis revealed that patients with IIM-associated ILD (IIM-ILD) had significantly higher levels of serum galectin-3 than those without IIM-ILD. In addition, patients with acute/subacute interstitial pneumonia had significantly higher levels of serum galectin-3 than those with chronic interstitial pneumonia. Furthermore, serum galectin-3 levels in IIM-ILD patients correlated with the radiological assessments of parenchymal lung involvement and treatment response. Immunohistochemical analysis revealed that galectin-3 was expressed in inflammatory cells of myositis and dermatitis sections, whereas in ILD sections, galectin-3 was expressed in interstitial fibrosis and inflammatory cells. Conclusion Galectin-3 may be involved in the pathogenesis of inflammatory and fibrotic conditions in IIM and can serve as a potential biomarker of disease activity, especially in patients with IIM-ILD.

Background: The occurrence of androgen-dependent prostate cancer mainly depends on prostate cancer stem cells. To reduce the risk of androgen-dependent prostate cancer, the direct elimination of prostate cancer stem cells is important, but an elimination strategy has not yet been established. A previous study showed that natural killer (NK) cells can preferentially target cancer stem cells in several solid tumors except prostate cancer. In this context, this study was undertaken to investigate if NK cells can selectively attack androgen-dependent prostate cancer stem cells. Methods: Prostate cancer stem-like cells were separated from an androgen-dependent prostate cancer cell line (LNCaP) using a three-dimensional culture system. LNCaP stem-like cells or LNCaP cells were co-cultured with human NK cells (KHYG-1) for 24–72 h, and cell viability was determined using the WST-8 method. The expression of each protein in the cell membrane was evaluated through FACS analysis, and mRNA levels were determined using real-time PCR. Results: KHYG-1 cells had more potent cytotoxicity against LNCaP stem-like cells than LNCaP cells, and the potency of the cytotoxicity was strongly related to the TRAIL/DR5 cell death pathway. Conclusion: NK cells can preferentially target prostate cancer stem-like cells via the TRAIL/DR5 pathway.

HER1-and HER2-targeted drugs are effective in cancer therapy, especially against lung, breast and colon malignancies; however, resistance of cancer cells to HER1-and HER2-targeted therapies is becoming a serious problem. The avidity/affinity constant (KA) and growth inhibitory effect of anti-HER3 rat monoclonal antibodies (mAb, Ab1∼Ab6) in the presence of therapeutic mAb or low-molecular-weight inhibitors against HER family proteins were analyzed by flow cytometry-based Scatchard plots (Splot) and cell proliferation assay. The KA of Ab3 and Ab6, but not Ab1 or Ab4, split into dual (high and low) modes of KA, and Ab6 exhibited greater anti-proliferative effects against LS-174T colon cancer cells in the presence of Pertuzumab (anti-HER2 mAb). A high KA by Ab6 and Ab6-mediated increased growth inhibition were observed against NCI–H1838 lung or BT474 breast cancer cells, respectively, in the presence of Panitumumab (anti-HER1 mAb) or Perutuzumab. A high KA by Ab6 and Ab6-mediated increased anti-proliferative effects against NCI–H1838 or BT474 were also respectively observed in the presence of Erlotinib (HER1 inhibitor) or Lapatinib (HER1/HER2 inhibitor). In HER1-knockout (KO) NCI–H1838, the reactivity and KA of Ab4 increased compared with in parent NCI–H1838. In HER1-KO or HER3-KO SW1116 colon cancer cells, dual modes of KA with Pertuzumab were noted, and the combination Ab6 and Pertuzumab promoted growth inhibition of HER1-KO, but not of parent SW1116.

In the present study, we hypothesized that habitual cigarette smoking attenuates endothelial function in the cerebral circulation as well as that of the peripheral circulation in young adults. To test this hypothesis, we measured cerebrovascular and peripheral flow‐mediated dilation (FMD) in young smokers and nonsmokers in the present study. Ten healthy nonsmokers and 10 smokers participated in the study. We measured blood velocity and diameter in the brachial artery and internal carotid artery (ICA) using Doppler ultrasound. We identified shear‐mediated dilation in the brachial artery and ICA by the percentage change in peak diameter during hyperemia stimulation (reactive hyperemia and hypercapnia). We measured the baseline diameter and the shear rate area under the curve from the onset of hyperemia to peak dilation in the brachial artery and ICA, finding the measurements of the smokers and those of the nonsmokers did not differ (p > .05). In contrast to brachial FMD (5.07 ± 1.79% vs. 7.92 ± 3.01%; smokers vs. nonsmokers, p = .019), FMD in the ICA was not attenuated in the smokers compared with that of the nonsmokers (5.46 ± 2.32% vs. 4.57 ± 2.70%; p = .442). These findings indicate that in young healthy smokers, cerebral endothelial function was preserved, and the response of cerebral endothelial function to smoking was different from that of peripheral vasculature., Peripheral FMD was lower in young healthy smokers than that in nonsmokers, indicating that habitual smoking attenuates peripheral endothelial function. In contrast, cerebral endothelial function was preserved in young healthy smokers. These findings suggest that the response of cerebral endothelial function to smoking is different from that of peripheral vasculature.

Background/aim EPH receptor A2 (EPHA2) is highly expressed in aggressive types of human cancer, and is expected to be an excellent target molecule for antibody treatments. In this study, we investigated the therapeutic potential of antibody to EPHA2 against melanoma in vitro. Materials and methods We generated three monoclonal antibodies (mAbs) to EPHA2 and examined cell-surface expression by flow cytometry. To investigate the ability to inhibit tumor cell migration therapy with mAbs to EPHA2, we performed a wound scratch assay and invasion assay. We investigated the therapeutic effects of immunotoxins consisting of toxin-conjugated EPHA2 mAbs. Results All human melanoma cell lines studied expressed EPHA2. Like natural ligand ephrin-A1, one of EPHA2 mAbs, SHM16, inhibited metastatic behavior of cells, such as migration and invasion. In addition, drastic growth inhibition and cytotoxicity were found using immunotoxin-conjugated SHM16. Conclusion These observations indicate a promising role for EPHA2 as a target in antibody treatments for melanoma, and demonstrate the potential therapeutic effects of an agonistic antibody to EPHA2.

Summary Background Scirrhous gastric cancer is associated with peritoneal dissemination and advanced lymph node metastasis from an early stage, and the prognosis is still poor. In this study, we aimed to analyze candidate molecules for targeted therapy of scirrhous gastric cancer. We searched for molecules/metabolic activity that might be predominantly expressed in a subpopulation of scirrhous gastric cancer cells and might function as cancer stem cell markers. Results For this purpose, we investigated the expression of various cell surface markers and of aldehyde dehydrogenase (ALDH) activity. These analyses showed that the scirrhous gastric cancer cell lines HSC-58 and HSC-44PE heterogeneously expressed CD13, while CD44, CDCP1, EpCAM and ABCG2 were expressed uniformly. Moreover, 10% of the total HSC-58 cell population expressed ALDH enzyme activity. A subpopulation of cells strongly positive for ALDH also expressed high levels of CD13, both of which are known as cancer stem cell markers. HSC-58 cells expressing high levels of CD13 showed lower sensitivity to a cancer drug cisplatin than cells with low levels of CD13. In contrast, CD13 -high subpopulation of HSC-58 was more sensitive to an aminopeptidase N inhibitor bestatin. In terms of antibody-drug therapy, anti-CD13-immunotoxin was highly cytotoxic towards HSC-58 cells and was more cytotoxic than anti-EpCAM-immunotoxin. Conclusion These data suggest that CD13 is a suitable cell surface candidate for targeted antibody-drug therapy of scirrhous gastric cancer.

Ergothioneine (ERG) is a natural antioxidant nutrient containing in edible golden oyster mushroom, Pleurotus cornucopiae (Tamogitake). In previous AACR meeting, we reported that administration of ERG rich water extracts of p. cornucopiae to tumor-bearing mice could suppress the growth of subcutaneous tumors and reduce the number of regulatory T cells (Treg) in lymph node and spleen. However, the molecular events inducing antitumor and immunomodulatory effects are still unknown. Our aim is to identify the mechanism of ERG in tumor cells which secrete various immunomodulatory cytokines. We cocultured human tumor cell lines with ERG and tested cytokine concentration in culture supernatants and expression of immune checkpoint molecules on tumors. ERG could significantly inhibit the production of VEGF, CCL2 and TGF-β1 which are important in not only angiogenesis and tumor invasion but also Treg induction. Importantly, ERG also reduced the expression of some immune checkpoint molecules on tumor cells and prolonged the survival of IL-2-activated lymphocytes in vitro. Overall, ERG rich water extract of p. citrinopileatus might inhibit the induction of Treg in tumor-bearing host via suppression of VEGF and TGF-β1, indicating a novel immunomodulatory antioxidant nutrient in mushrooms Citation Format: Kazunori Kato, Katsuhiro Kuroki, Yui Shimizu. Anti-oxidant amino acid L-ergothioneine exhibits immunomodulatory effect by regulating immunosuppressive cytokine production of tumor cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1020.

Despite aggressive surgical resections and combinatorial chemoradiations, certain highly malignant populations of tumor cells resurrect and metastasize. Mixed-grade cancer cells fail to respond to standard-of-care therapies by developing intrinsic chemoresistance and subsequently result in tumor relapse. Macroautophagy is a membrane trafficking process that underlies drug resistance and tumorigenesis in most breast cancers. Manipulating cellular homeostasis by a combinatorial nanotherapeutic model, one can evaluate the crosstalk between type I and type II cell death and decipher the fate of cancer therapy. Here, we present a multi-strategic approach in cancer targeting to mitigate the autophagic flux with subcellular toxicity via lysosome permeation, accompanied by mitochondrial perturbation and apoptosis. In this way, a nanoformulation is developed with a unique blend of a lysosomotropic agent, an immunomodulating sulfated-polysaccharide, an adjuvant chemotherapeutic agent, and a monoclonal antibody as a broad-spectrum complex for combinatorial nanotherapy of all breast cancers. To the best of our knowledge, this manuscript illustrates for the first time the applications of advanced microscopic techniques such as electron tomography, three-dimensional rendering and segmentation of subcellular interactions, and fate of the multifunctional therapeutic gold nanocages specifically targeted toward breast cancer cells.

Nanoscience and nanotechnology utilize nanoparticles to provide several remarkable applications in biomedical fields such as bio imaging, diagnosis of several diseases, and efficient drug delivery. Multifunctional magnetic nanoparticles (MNPs) are a significant type of nanomaterials that are capable of developing therapeutic as well as diagnostic (theragnostic) applications. Multifunctional hybrid magnetic nanoparticles, as efficient theragnostic representatives, have been developed. The hybrid magnetic nanoparticles (HMNP)-Au-MNPs have been assimilated into PLGA NPs along with two potent chemotherapeutic agents (curcumin and gemcitabine). Delivering chemotherapeutics into cancer cells with multiple targeting moieties is an attractive area in cancer therapeutics. In this study, the nanoconjugates have been tailored to target the pancreatic and breast cancer cells by attaching three specific targeting ligands (folate, transferrin and AS1411 aptamer). Moreover, we report a nanoparticle-based delivery system that can deliver therapeutic cargos to the cancer cells and can exert its lethality by three distinctive modes (chemotherapeutic, photothermal and magnetic hyperthermia) either independently or by the synergistic action of drug and hyperthermia. Overall our results display an 'all in one' approach with the same nanoformulation for the combined destruction of cancer cells. The developed nanoformulation was highly versatile in terms of the choice of implementing lethality, depending on the cancer type. Refereed/Peer-reviewed

Superparamagnetic iron oxide nanoparticles (SPIONs) employed theragnostic work in cancer therapy exploited its inherent magnetic properties that can be utilized for drug targeting and for drug delivery applications. To attain theragnostic properties, SPION and curcumin encapsulated HER2 targeted PLGA (HER2-CUR-SPION-PLGA) nano-formulation was developed by solvent evaporation technique (CUR-SPION-PLGA NPs). The physicochemical properties of nanoconjugate were characterized using various instruments such as TEM, SEM, AFM, XPS, and VSM etc. The uptake of NPs was studied in pancreatic cancer cells (PANC-1 and MIA PaCa-2) and normal fibroblast cells (L929) by confocal microscopy and flowcytometry analysis. The anticancer potential of the NPs in the absence and presence of an external magnet (magnetic targeting property) was studied. The effect of curcumin on mitochondrial membrane potential integrity leading to apoptosis was also determined. The conjugation of HER2 to nanoconjugate for active magnetic targeting for the co-delivery of curcumin and MNPs enhanced synergistic therapeutic index. HER2-CUR-SPION-PLGA NPs displayed promising potential anticancer activity by destroying pancreatic cancer cells. Refereed/Peer-reviewed

CD146 (MCAM) expressed on not only vascular endothelial and smooth muscle cells but also various malignant tumor cells. It has been shown that overexpression of CD146 predicts poor prognosis of solid tumor. Several reports by using transfectants or gene silencing technique have shown that CD146 is involved in cell adhesion and inflammatory cell migration. However, contribution of CD146 in tumor metastasis and antitumor immunity are still unknown. To investigate the effect of CD146 on tumor metastatic properties, we establish two subclones from DLD-1 (human colon adenocarcinoma cells) expressing CD146 or not. Both cell lines express epithelial tumor marker EpCAM and TROP2 equally, while CD146 (+) DLD-1 could only express CD44 variant v8–v10 and EphA2. Using metabolic assay, we found enhanced metabolism and decreased lactate production in CD146 (+) DLD-1. Consist with the morphological changes to mesenchymal features CD146 (+) DLD-1, the expression of E-cadherin and claudin-3 in tight junction were decreased in CD146 (+) DLD-1. In addition, CD146 (+) DLD-1 significantly increased the production of VEGF and the induction of various genes (slug, twist and zeb1) which are involved in epithelial to mesenchymal transition (EMT). Interestingly, the expression of immune checkpoint molecules including B7-H1, B7-DC (both PD-1 ligands), CD155 and IDO were up-regulated in CD146 (+) DLD-1 inducing the resistance of cytotoxic effect of activated lymphocytes. These results indicate that CD146 (+) DLD-1 strongly induces immune escape mechanism, thus demonstrating a new relationship between CD146 and EMT and anti-tumor immunity, which would be a new treatment and diagnostic target for immunotherapy.

Establishment of a system that allows selective drug delivery and gene silencing to a tumor is expected to enable targeted therapy. We constructed a genetically modified adenovirus incorporating an IgG Fc-binding motif from the Staphylococcus protein A, Z33 (Adv-FZ33). By cross-linking the Adv-FZ33 virus and the surface antigen molecules with the targeting monoclonal antibodies (mAbs), we attained highly enhanced gene deliveries into the respective antigen-positive cancer cells. Therefore, we aimed to establish a systematic screening method to search for antibody and cell surface target candidates that would provide highly selective anti-cancer reagents to malignant tumors. Using an Adv-FZ33, hybridoma libraries producing a variety of mAbs for human pancreatic, prostate, lung or ovarian carcinoma cells were screened, and we were able to selectively obtain several mAbs which had potent high affinity and recognized antigens of high structure. Within these mAbs, we have identified tumor cell target molecules including not only carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), prostate specific membrane antigen (PSMA) but also novel tumor surface target molecules such as phosphatidic acid phosphatase type 2a (PAP2a) and interleukin-13 receptor variant α2 (IL-13Rα2) as tumor antigens. Overall, these results indicate that this type of inductive method approach is a reliable strategy for screening in antibody therapy on par with antibody-dependent drug-delivery system.

Background Multiple factors affect the in vivo antitumor activity of antibody-based therapeutics; however, the influence of cell surface retention on antibody-dependent cellular cytotoxicity (ADCC) is not fully understood. Here we evaluated the importance of cell surface antibody retention in antitumor activity mediated by ADCC in vivo. Materials and methods Two mAbs against tumor-associated calcium signal transducer 2 (TACSTD2/TROP2), AR47A6.4.2 and Pr1E11, were used. Antitumor activities against BxPC3 and Colo205 cells were investigated through in vitro and in vivo assays. Results Pr1E11 showed better cell surface retention than AR47A6.4.2 in vitro although Pr1E11 and AR47A6.4.2 showed equivalent ADCC activity. Complement-dependent cytotoxicity and antiproliferative activity were not observed for either antibody. Pr1E11 exhibited higher antitumor activity than AR47A6.4.2 in vivo. Conclusion Our results suggest that high cell surface retention can result in potent ADCC activity in vivo. This observation could provide novel insight into how effectively screen for antibodies with strong in vivo antitumor activity.

CD146, a transmembrane glycoprotein member of the immunoglobulin superfamily, acts as an adhesion molecule that helps maintain the cell monolayer. Human endothelial cells expressing CD146 are involved in angiogenesis and inflammation. Recently, we developed a sandwich ELISA for detecting soluble CD146 (sCD146) in human serum specimens. The aim of this study is to determine serum levels of sCD146 in patients with systemic sclerosis (SSc) and to examine the relationship between sCD146 levels and clinical manifestations. We quantified serum sCD146 levels in 47 serum samples from patients fulfilling criteria for SSc, 23 serum samples from patients fulfilling criteria for rheumatoid arthritis (RA), and 25 healthy controls. We also investigated the relationship between sCD146 levels and various clinical characteristics with SSc patients. Levels of sCD146 were significantly higher in the 47 patients with SSc than in the 25 healthy controls and 23 patients with RA (12.50 vs. 6.91 vs. 9.95 ng/ml; p

Natural killer (NK) cells play a central role as innate immune effector cells to virus infected cells and tumor cells. The standard assay to evaluate NK activity is 51Cr release assay using radio-labeled K562 cells, however, new methods using fluorescence-labeled targets or flow cytometry to examine degranulation markers have been developed recently. In addition to cytotoxicity, NK cells enable to produce various cytokines to regulate the adaptive immune responses in infection, inflammation and cancer. Here we generated nanoparticles conjugated with various antibody for NK receptors and examined to stimulate human whole blood with these nanoparticles instead of K562 target cells. We found that various cytokines such as IFN-γ, IL-6, IL-1β and CCL2 were produced by NK cells in whole blood by anti-NKp30-conjugated nanoparticles in dose-dependent manner more than by anti-NKp46 or anti-NKG2D nanoparticles. Production of IFN-γ was detected within 4 hours and reached maximum level at 24 hours after stimulation. IFN-γ production was relatively high at incubation temperature of 37°C, but that was significantly decreased over 39°C. Importantly, we could evaluate the reproducible cytokine production in same donors at another blood collection date because of the stable stimulus of nanoparticles and less variation data by human manipulation for lymphocyte separation. Additionally, cytokine secretion did not correlate with NK cell-mediated lysis of K562 target cells and NK cell numbers in whole blood. These data indicated that this method using NKp30 nanoparticles might be useful for measurement of individual differences of NK function especially correlated in adaptive immune response and inflammation.

Background Neuron-selective gene transfer is an attractive therapeutic strategy for neurological disorders. However, optimal targets and gene delivery systems remain to be determined. Methods Following immunization of mice with PC12 cells, hybridomas were screened by β-Gal reporter gene assay using FZ33 fiber-modified adenovirus vectors. Subsequently, the efficacy and specificity of monoclonal antibody (mAb)-mediated gene transfer via FZ33 and FdZ adenovirus vectors were evaluated by flow cytometry, chemiluminescent β-Gal reporter gene assay, and immunocytochemistry. Finally, the antigen recognized by the mAb was identified by mass spectrometry and transfection analysis. Results A hybridoma clone 6E3 producing monoclonal antibody, mAb6E3, was screened. Flow cytometry, chemiluminescent β-Gal reporter gene assay, and immunocytochemistry with mAb6E3 and the fiber mutant adenovirus demonstrated efficient gene transfer into the PC12 cells. Treatment of neuron–glia cocultures with mAb6E3 and FdZ adenovirus resulted in neuron-selective gene transfer. Immunohistochemical images of rat spinal cord tissue showed that mAb6E3 reacts specifically with neurons. Finally, Na,K-ATPase β1 was identified as the antigen of mAb6E3. Conclusions Hybridoma screening using FZ33 fiber-modified adenovirus vectors serves as an efficient approach to detect antigens in mAb-targeted gene transfer. Neuronal tropism in the central nervous system through mAb6E3 represents an important initial step towards neuron-selective gene transfer in the treatment of local neurological disorders, such as spinal cord injury. Copyright © 2008 John Wiley & Sons, Ltd.

In this study, we present evidence that gene transfer of the CD40-ligand (CD154) into human immature dendritic cells (DC) imparts direct antitumor effects on tumor cells. DC infected with adenovirus directed to express human CD154 on the cell surface (CD154-DC) elicited significantly higher levels of immune accessory molecules commonly found on mature DC. We found that co-cultivation with a human squamous cell carcinoma cell line (OSC-70) with CD154-DC significantly inhibited cell growth. We further demonstrate that OSC-70 cells stimulated with CD154-DC were more susceptible to apoptosis via TNF-related apoptosis inducing ligand (TRAIL). Importantly, tumor cells co-cultured with CD154-DC in transwell plates expressed upregulated cell surface TRAIL-R2. CD154-DC produced higher levels of interferon (IFN)-gamma, IL-12p70 and soluble CD154, but the ability of CD154-DC to inhibit tumor cell growth was significantly abrogated by a neutralizing antibody to IFN-gamma, indicating that this was mainly mediated by IFN-gamma. Furthermore, intratumoral injection of CD154-DC significantly suppressed OSC-70 tumor growth in a xenograft model. Overall, these results reveal that CD154-DC have potential as an anti-cancer therapy by producing IFN-gamma to arrest adjacent tumor cell growth and increase the susceptibility of apoptosis via TRAIL.

CD154 (CD40-ligand) is a critical transmembrane molecule with potent immune-stimulatory properties that is used in clinical applications of gene therapy for leukemia and lymphoma. However, CD154 is cleaved into a soluble form, and high levels of sCD154 contribute to systemic inflammatory and cardiovascular diseases, suggesting a deleterious side effect of CD154 gene therapy. In this study, we engineered noncleavable mutants of human CD154 with point mutations to develop a potentially less toxic molecule in vivo. In contrast to wild-type CD154 (CD154-WT) subsequently released as sCD154, both mutants CD154-M3 and CD154-M4 were resistant to cleavage in tumor cells. Also, CD40-expressing leukemia B cells transfected with CD154-M3 mutant were highly effective stimulators in a mixed lymphocyte-leukemia reaction, indicating that CD154-M3 mutant did not lose biologic activity. In mice transplanted with tumors expressing CD154-WT, we found increased plasma levels of human sCD154 followed by various systemic inflammatory reactions such as glomerulonephritis and an increased number of infiltrating mononuclear cells in the liver. However, CD154-M3 mutant did not induce any systemic inflammatory effects in vivo. As such, the noncleavable mutant of CD154 is fully capable of inducing the immune response with less toxic properties and is a useful tool for CD154 immune gene therapy.

Objective Chronic lymphocytic leukemia (CLL) B cells from most patients express both membrane-bound CD27 (mCD27) and soluble CD27 (sCD27). Expression of sCD27 inhibits CD27-dependent T-cell or CLL-cell activation mediated by its ligand, CD70. In this study, we evaluated whether protease inhibitors could inhibit the release of sCD27 from CLL cells and enhance T-cell activation mediated by CD27-CD70 interaction. Methods CLL cells exposed to hydroxamic acid-based matrix metalloprotease (MMP) inhibitors were evaluated for the release of sCD27 by sandwich enzyme-linked immunosorbent assay and immunoprecipitation. We examined for phenotypic changes in CLL cells treated with MMP inhibitors by flow cytometry and T-cell activation by CLL cells was assessed by [ 3 H] thymidine incorporation assay and the production of interferon-gamma. Results Treatment of CLL cells with MMP inhibitors blocked the release of sCD27 to the culture supernatant. In contrast, a non–hydroxamic acid control compound or inhibitors of other proteases, including serine, cysteine, and aspartyl proteases, were ineffective. Furthermore, CLL cells treated with MMP inhibitors expressed significantly higher levels of accessory molecules, such as CD54, CD80, and CD95. Consistent with such changes, we found that CLL cells treated with MMP inhibitors, but not control treated cells, could stimulate allogeneic and autologous T cells in mixed lymphocyte reactions. Conclusion These data reveal that metalloprotease inhibitors can block production of sCD27, which can interfere with mCD27-CD70 interactions that induce expression of immune costimulatory molecules on CLL B cells. Conceivably, treatment of CLL cells with metalloprotease inhibitors may enhance their potential for stimulating cellular immune recognition of leukemia-associated antigens.

The thermal conductivity of the surface layer of engineering materials changes in the early stages of material degradation due to the appearance of micro-cracks. A new method for evaluating and assessing the degree of degradation of a material using this change is proposed herein. The influence of the micro-cracked layer on the temperature response as measured by a thermophysical handy tester was theoretically examined. By defining a thermal degradation parameter, the amount of degradation of various materials was evaluated. In order to verify this theory, fatigue tests using metallic materials were conducted. Comparisons before and after the fatigue tests were then made to establish the correlation between the density of micro-cracks and the decrease in thermal conductivity. As a result, it was ascertained that a thermal degradation parameter can easily be estimated from a temperature response curve obtained using the tester. © 2007 Wiley Periodicals, Inc. Heat Trans Asian Res, 36(8): 501–512, 2007; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/htj.20178

BACKGROUND Focusing on Adv-FZ33, a modified adenovirus in which a synthetic 33-amino-acid immunoglobulin G-binding domain was inserted into the adenoviral fiber protein, we tried to identify suitable target molecules for prostate cancer-specific gene therapy. METHODS Hybridomas were established from mice immunized with prostate cancer cell lines. The hybridomas were screened using Adv-FZ33 to create monoclonal antibodies (mAbs) that induced high gene transfer efficiency for PC-3 cells. Furthermore, we identified target antigens of the mAbs by immunoprecipitation and mass spectrometry, and investigated the expression of target molecules by flow cytometry and immunocytochemistry. RESULTS Using Adv-FZ33, we established four different mouse mAbs that increased transduction efficiency for PC-3. The target antigens identified were Ep-CAM, CD155, HAI-1, and Na,K-ATPase β1. These antigens were expressed in several cancer cell lines, including prostate cancer. Human prostatic myofibroblast cells lacked expression of Ep-CAM and HAI-1. CONCLUSIONS We established anti-Ep-CAM mAb and anti- HAI-1 mAbs. Gene transduction via Ep-CAM and HAI-1 may be a novel strategy for treatment of prostate cancer. Prostate 67: 1163–1173, 2007. © 2007 Wiley-Liss, Inc.

筆者らは，プロテインA由来の33個ペプチド配列をファイバー部に組み込んだアデノウイルス（Adv-FZ33）を作製しその生物活性を検討してきた．さまざまなヒト腫瘍標的細胞と結合し，このファイバー変異型アデノウイルスを介した遺伝子導入効率を増強できる標的化抗体の網羅的な樹立法の開発に成功した．

TROP-2 is a type Ⅰ transmembrane glycoprotein that is highly expressed in various epithelial cancer cells, and its increased expression correlates with poor prognosis. Although several anti-TROP-2 antibodies have been described, they were found unsuitable for antitumor therapy use in vivo as naked antibodies. In this study, we established a novel anti-TROP-2 antibody, designated Pr1E11, from mice immunized with primary prostate cancer cells. Antibody screening was based on the infection activity of Adv-LacZ-FZ33, which displays an immunoglobulin G binding domain in the adenoviral fiber protein. We found that Pr1E11 specifically binds to TROP-2 with high affinity and recognizes diverse epithelial cancer cell lines and primary pancreatic cancer tissues. Epitope analysis using TROP-2 deletion mutants revealed that binding site of Pr1E11 is a cysteine-rich domain, a unique epitope compared with other available anti-TROP-2 antibodies. In addition, Pr1E11 exhibited low internalization activity, which may make it suitable for naked antibody therapeutics. Our results suggest that Pr1E11 may stimulate different biological activities from other anti-TROP-2 antibodies based on its unique binding epitope, and is a potential candidate for naked antibody therapeutics for various epithelial cancer treatments.

RNA interference and the therapeutic applications using small interfering RNA was discovered more than 10 years ago and currently is used in various applications including in therapeutic field. However the research in this field is still in its infancy. Many challenges like safe delivery of targeted siRNA to nucleus and cytosol of cancerous cells without compromising the activity of siRNA needs to be addressed. We have overcome this hurdle with the help of nanotechnology using PLGA hollow nanoparticles (PLGAHNPs) and suppressing the oncogene of MYC transcription factors by using anti myc-siRNAs in human cancer cell lines. siRNA was encapsulated in PLGAHNPs. These spherical PLGAHNPs of size 70 nm had high efficiency of gene release at pH 4.2 under in vitro conditions. Cell penetrating peptide (CPP)- Tat peptide (TAT) and peptide nucleic acidnucleolus localizing signal (PNA-NLS) was used for siRNA delivery without affecting the therapeutic activity of siRNA. The siRNA duplex was prepared using T7 polymerase and double stranded DNA through in vitro transcription. Incubation of the siRNA encapsulated PLGAHNPs functionalized with TAT and PNA-NLS (TAT-siRNA-PNA-PLGAHNPs-siRNA) with cancer cells resulted in reduced cell proliferation. A downregulation of gene expression by 90% was observed even with low concentration of siRNA. We found complete arrest of cell division which was mediated by downregulation of MYC expression.

Purpose: A major problem when using the adenoviral vectors for gene therapy applications is thought to be related to low transduction efficiency in cancer cells or to side effects in normal cells. There is an urgent requirement to improve the specificity of gene delivery in the context of cancer gene therapy.Experimental Design: We constructed a genetically modified adenovirus incorporating an IgG Fc-binding motif from the Staphylococcus protein A, Z33, within the HI loop (Adv-FZ33). A remarkable degree of targeted gene delivery to gastric cancer cells was obtained with Adv-FZ33 with the fully human anti–carcinoembryonic antigen (CEA) monoclonal antibody, C2-45.Results: In vitro LacZ or EGFP gene expression after Adv-FZ33 infection via C2-45 was 20 times higher than control monoclonal antibody in MKN-45 at 1,000 viral particles/cell. We generated Ax3CAUP-FZ33 (UP-FZ33), which is an Adv-FZ33 derivative vector expressing a therapeutic gene (i.e., Escherichia coli uracil phosphoribosyltransferase), which converts 5-fluorouracil (5-FU) directly to 5-fluoro-UMP. UP-FZ33 with C2-45 enhanced the cytotoxicity of 5-FU by 10.5-fold in terms of IC50 against MKN-45 compared with control IgG4. In a nude mouse peritoneal dissemination model, tumor growth in mice treated with UP-FZ33/C2-45/5-FU was significantly suppressed, and tumor volumes were less than one-fourth of those of the control IgG4 group (P < 0.05). The median survival time of the UP-FZ33/C2-45/5-FU group was significantly longer than those treated with PBS or 5-FU only (P < 0.01).Conclusions: These data suggest that CEA-targeted FZ33 mutant adenovirus-mediated gene delivery offers a strong and selective therapeutic modality against CEA-producing cancers.

BACKGROUND Although neuroendocrine (NE) cells in prostate cancer have been speculated to accelerate the growth and progression of surrounding cancer cells, the evidence is as yet inconclusive. We investigated the effect of an NE allograft (NE-10) and its cell line, NE-CS, which were established from the prostate of the LPB-Tag 12T-10 transgenic mouse, on human prostate cancer cell line LNCaP. METHODS The proliferation and pulmonary metastasis of LNCaP xenografts in athymic mice with and without NE-10 allografts were evaluated. Boyden chamber assay and microarray analysis were performed to investigate changes in invasion/migration and mRNA of LNCaP cells under the influence of the NE cells, respectively. RESULTS NE-10 did not influence the proliferation of LNCaP. The pulmonary metastasis of LNCaP with NE-10 significantly increased compared to mice without it. The NE-CS cells accelerated the in vitro invasion/migration of adenocarcinoma cells. Increased expression of mRNA of gelsolin was observed in LNCaP cells incubated with the supernatant of NE-CS cells. CONCLUSIONS The NE-10 allograft promotes pulmonary metastasis of subcutaneously inoculated LNCaP cells by facilitating cell invasion. Secretions from NE cells upregulate the expression of gelsolin, which is an actin-binding protein, resulting in acceleration of the migration of LNCaP cells. © 2005 Wiley-Liss, Inc.

This paper presents the applicability of a thermophysical handy tester for quality inspection and diagnostic technique such as in situ measurement of polymeric resin products. Influence of crystallinity known as a rating factor of quality, or filler concentration upon thermal conductivity is determined for the case of unsaturated polyester resin products by using the tester. Consequently, good correlations between the thermal conductivity and the crystallinity or the filler concentration are certified. The variation or the distribution of thermal conductivity of products molded under various conditions can be detected nondestructively using the tester. As an example, the thermal conductivity distribution around the forming gate is demonstrated, indicating the density-uniformity of the resin product. © 2006 Wiley Periodicals, Inc. Heat Trans Asian Res, 35(6): 421–433, 2006; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/htj.20124

In this study, we developed novel double-membranous non-viral gene delivery system modified with SV-40 T antigen-derived nuclear localization signal (NLS-DMEND) for delivery of luciferase plasmid DNA to nucleus of non-dividing mouse bone marrow-derived dendritic cells (BMDC). Intracellular trafficking and gene expression of NLS-DMEND in the BMDC were evaluated. Condensed DNA was observed in the nucleus by confocal laser scanning microscopy, and the NLS-DMEND induced significant luciferase activity in the BMDC. It was suggested that the condensed DNA particle transferred into nucleus via energy dependent manner, since the nuclear transfer was inhibited by metabolic inhibitors. In conclusion, condensed plasmid DNA was delivered into the nucleus of non-dividing BMDC by NLS-DMEND.

Background— Apoptosis is an important cause of early graft loss after heart transplantation. Bcl-xL was reported to protect the heart against normothermic ischemia and reperfusion injury. In this study, we determined whether overexpression of Bcl-xL could inhibit tissue injury resulting from prolonged cold preservation followed by warm reperfusion of heart transplants. Methods and Results— Lewis rat hearts were transduced with an adenovirus vector harboring Bcl-xL cDNA (AxCAhBclxL) 4 days before collection of tissue. After preservation in University of Wisconsin solution at 4°C for 24 hours, the heart was either perfused with a Langendorff device ex vivo or used for heterotopic heart transplantation in vivo. Bcl-xL gene transfer significantly reduced the infarct size (23.0±2.6% versus 47.7±7.0% in saline control and 48.6±6.1% in vector control, P P c release from the mitochondria; it also significantly decreased cardiac cell apoptosis and improved graft survival rate after long cold preservation, followed by warm reperfusion. Conclusions— Bcl-xL gene transfer inhibited the translocation of Bax and prolonged the cold preservation time of cardiac transplants. This may be a potential therapeutic method in clinical practice.