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ObjectiveStromal barriers, such as the abundant desmoplastic stroma that is characteristic of pancreatic ductal adenocarcinoma (PDAC), can block the delivery and decrease the tumour-penetrating ability of therapeutics such as tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), which can selectively induce cancer cell apoptosis. This study aimed to develop a TRAIL-based nanotherapy that not only eliminated the extracellular matrix barrier to increase TRAIL delivery into tumours but also blocked antiapoptotic mechanisms to overcome TRAIL resistance in PDAC.DesignNitric oxide (NO) plays a role in preventing tissue desmoplasia and could thus be delivered to disrupt the stromal barrier and improve TRAIL delivery in PDAC. We applied an in vitro–in vivo combinatorial phage display technique to identify novel peptide ligands to target the desmoplastic stroma in both murine and human orthotopic PDAC. We then constructed a stroma-targeted nanogel modified with phage display-identified tumour stroma-targeting peptides to co-deliver NO and TRAIL to PDAC and examined the anticancer effect in three-dimensional spheroid cultures in vitro and in orthotopic PDAC models in vivo.ResultsThe delivery of NO to the PDAC tumour stroma resulted in reprogramming of activated pancreatic stellate cells, alleviation of tumour desmoplasia and downregulation of antiapoptotic BCL-2 protein expression, thereby facilitating tumour penetration by TRAIL and substantially enhancing the antitumour efficacy of TRAIL therapy.ConclusionThe co-delivery of TRAIL and NO by a stroma-targeted nanogel that remodels the fibrotic tumour microenvironment and suppresses tumour growth has the potential to be translated into a safe and promising treatment for PDAC.

The globo-series glycosphingolipids (SSEA3, SSEA4, and Globo H) were shown to express in many cancers selectively, and a combination of anti-SSEA4 and anti-Globo H antibodies was able to suppress tumor growth in mice inoculated with breast cancer cell lines. To further understand the effect, we focused on the combined effect of the two antibodies in target binding and antibody-dependent cellular cytotoxicity (ADCC) in vitro. Here, we report that the binding of anti-Globo H antibody (VK9) to MDA-MB231 breast cancer cells was influenced by anti-SSEA4 antibody (MC813-70), and a combination of both antibodies induced a similar effect as did anti-SSEA4 antibodies alone in a reporter-based ADCC assay, indicating that SSEA4 is a major target in breast cancer due to its higher expression than Globo H. Furthermore, we showed that a homogeneous anti-SSEA4 antibody (chMC813-70-SCT) designed to maximize the ADCC activity can be used to isolate a subpopulation of natural killer (NK) cells that exhibit an ∼23% increase in killing the target cells as compared to the unseparated NK cells. These findings can be used to predict a therapy outcome based on the expression levels of antigens and evaluate therapeutic antibody development.

Background With the continuous emergence of new SARS-CoV-2 variants that feature increased transmission and immune escape, there is an urgent demand for a better vaccine design that will provide broader neutralizing efficacy. Methods We report an mRNA-based vaccine using an engineered “hybrid” receptor binding domain (RBD) that contains all 16 point-mutations shown in the currently prevailing Omicron and Delta variants. Results A booster dose of hybrid vaccine in mice previously immunized with wild-type RBD vaccine induced high titers of broadly neutralizing antibodies against all tested SARS-CoV-2 variants of concern (VOCs). In naïve mice, hybrid vaccine generated strong Omicron-specific neutralizing antibodies as well as low but significant titers against other VOCs. Hybrid vaccine also elicited CD8+/IFN-γ+ T cell responses against a conserved T cell epitope present in wild type and all VOCs. Conclusions These results demonstrate that inclusion of different antigenic mutations from various SARS-CoV-2 variants is a feasible approach to develop cross-protective vaccines.

Throughout the COVID-19 pandemic, many prophylactic and therapeutic drugs have been evaluated and introduced. Among these treatments, monoclonal antibodies (mAbs) that bind to and neutralize SARS-CoV-2 virus have been applied as complementary and alternative treatments to vaccines. Although different methodologies have been utilized to produce mAbs, traditional hybridoma fusion technology is still commonly used for this purpose due to its unmatched performance record. In this study, we coupled the hybridoma fusion strategy with mRNA-lipid nanoparticle (LNP) immunization. This time-saving approach can circumvent biological and technical hurdles, such as difficult to express membrane proteins, antigen instability, and the lack of posttranslational modifications on recombinant antigens. We used mRNA-LNP immunization and hybridoma fusion technology to generate mAbs against the receptor binding domain (RBD) of SARS-CoV-2 spike (S) protein. Compared with traditional protein-based immunization approaches, inoculation of mice with RBD mRNA-LNP induced higher titers of serum antibodies. In addition, the mAbs we obtained can bind to SARS-CoV-2 RBDs from several variants. Notably, RBD-mAb-3 displayed particularly high binding affinities and neutralizing potencies against both Alpha and Delta variants. In addition to introducing specific mAbs against SARS-CoV-2, our data generally demonstrate that mRNA-LNP immunization may be useful to quickly generate highly functional mAbs against emerging infectious diseases.

Ovarian cancer has a high recurrence rate after platinum‐based chemotherapy. To improve the treatment of ovarian cancer and identify ovarian cancer‐specific antibodies, we immunized mice with the human ovarian carcinoma cell line, SKOV‐3, and generated hybridoma clones. Several rounds of screening yielded 30 monoclonal antibodies (mAbs) with no cross‐reactivity to normal cells. Among these mAbs, OV‐Ab 30‐7 was found to target integrin α3 and upregulate p53 and p21, while stimulating the apoptosis of cancer cells. We further found that binding of integrin α3 by OV‐Ab 30‐7 impaired laminin‐induced focal adhesion kinase phosphorylation. The mAb alone or in combination with carboplatin and paclitaxel inhibited tumor progression and prolonged survival of tumor‐bearing mice. Moreover, immunohistochemical staining of ovarian patient specimens revealed higher levels of integrin α3 in cancer cells compared with normal cells. By querying online clinical databases, we found that elevated ITGA3 expression in ovarian cancer is associated with poor prognosis. Taken together, our data suggest that the novel mAb, OV‐Ab 30‐7, may be considered as a potential therapeutic for ovarian cancer., The novel mAb, OV‐Ab 30‐7, can induce ovarian cancer cell apoptosis, and blockage integrin‐laminin signaling, and may be considered as a potential therapeutic for ovarian cancer.

Pancreatic cancer is usually asymptomatic in the early stages; the 5-y survival rate is around 9%; and there is a lack of effective treatment. Here we show that SSEA-4 is more expressed in all pancreatic cancer cell lines examined but not detectable in normal pancreatic cells; and high expression of SSEA-4 or the key enzymes B3GALT5 + ST3GAL2 associated with SSEA-4 biosynthesis significantly lowers the overall survival rate. To evaluate potential new treatments for pancreatic cancer, homogeneous antibodies with a well-defined Fc glycan for optimal effector functions and CAR-T cells with scFv construct designed to target SSEA-4 were shown highly effective against pancreatic cancer in vitro and in vivo. This was further supported by the finding that a subpopulation of natural killer (NK) cells isolated by the homogeneous antibody exhibited enhancement in cancer-cell killing activity compared to the unseparated NK cells. These results indicate that targeting SSEA-4 by homologous antibodies or CAR-T strategies can effectively inhibit cancer growth, suggesting SSEA-4 as a potential immunotherapy target for treating pancreatic disease.

Upregulation of interleukin-17 receptor B (IL-17RB) is known to be oncogenic, while other IL-17 receptors and ligands are generally involved in pro-inflammatory pathways. We identify a mouse neutralizing monoclonal antibody (mAb) D9, which blocks the IL-17RB/IL-17B pathway and inhibits pancreatic tumorigenesis in an orthotopic mouse model. The X-ray crystal structure of the IL-17RB ectodomain in complex with its neutralizing antibody D9 shows that D9 binds to a predicted ligand binding interface and engages with the A'-A loop of IL-17RB fibronectin III domain 1 in a unique conformational state. This structure also provides important paratope information to guide the design of antibody humanization and affinity maturation of D9, resulting in a humanized 1B12 antibody with marginal affinity loss and effective neutralization of IL-17B/IL-17RB signaling to impede tumorigenesis in a mouse xenograft model.

Since the pandemic of COVID-19 has intensely struck human society, small animal model for this infectious disease is in urgent need for basic and pharmaceutical research. Although several COVID-19 animal models have been identified, many of them show either minimal or inadequate pathophysiology after SARS-CoV-2 challenge. Here, we describe a new and versatile strategy to rapidly establish a mouse model for emerging infectious diseases in one month by multi-route, multi-serotype transduction with recombinant adeno-associated virus (AAV) vectors expressing viral receptor. In this study, the proposed approach enables profound and enduring systemic expression of SARS-CoV-2-receptor hACE2 in wild-type mice and renders them vulnerable to SARS-CoV-2 infection. Upon virus challenge, generated AAV/hACE2 mice showed pathophysiology closely mimicking the patients with severe COVID-19. The efficacy of a novel therapeutic antibody cocktail RBD-chAbs for COVID-19 was tested and confirmed by using this AAV/hACE2 mouse model, further demonstrating its successful application in drug development., Author summary Upon the emergence of new infectious disease, animal model becomes a pivotal tool for study of disease mechanism and development of therapeutics. In this study, we propose a versatile approach that allows rapid generation of mouse model for novel infectious disease once the receptor of the pathogen is identified. We demonstrated this approach by generating a mouse model for COVID-19 in a month’s time. These mice were capable of recapitulating severe COVID-19 in patients, and successfully applied in the development of a therapeutic antibody cocktail for the disease. This not only suggests the usefulness of this mouse model for the research on COVID-19, but also exhibit the utility of the proposed approach for establishing animal model for infectious disease.

Vascular endothelial growth factor receptor 2 (VEGFR2) is highly expressed in tumor‐associated endothelial cells, where it modulates tumor‐promoting angiogenesis, and it is also found on the surface of tumor cells. Currently, there are no Ab therapeutics targeting VEGFR2 approved for the treatment of prostate cancer or leukemia. Therefore, development of novel efficacious anti‐VEGFR2 Abs will benefit cancer patients. We used the Institute of Cellular and Organismic Biology human Ab library and affinity maturation to develop a fully human Ab, anti‐VEGFR2‐AF, which shows excellent VEGFR2 binding activity. Anti‐VEGFR2‐AF bound Ig‐like domain 3 of VEGFR2 extracellular region to disrupt the interaction between VEGF‐A and VEGFR2, neutralizing downstream signaling of the receptor. Moreover, anti‐VEGFR2‐AF inhibited capillary structure formation and exerted Ab‐dependent cell‐mediated cytotoxicity and complement‐dependent cytotoxicity in vitro. We found that VEGFR2 is expressed in PC‐3 human prostate cancer cell line and associated with malignancy and metastasis of human prostate cancer. In a PC‐3 xenograft mouse model, treatment with anti‐VEGFR2‐AF repressed tumor growth and angiogenesis as effectively and safely as US FDA‐approved anti‐VEGFR2 therapeutic, ramucirumab. We also report for the first time that addition of anti‐VEGFR2 Ab can enhance the efficacy of docetaxel in the treatment of a prostate cancer mouse model. In HL‐60 human leukemia‐xenografted mice, anti‐VEGFR2‐AF showed better efficacy than ramucirumab with prolonged survival and reduced metastasis of leukemia cells to ovaries and lymph nodes. Our findings suggest that anti‐VEGFR2‐AF has strong potential as a cancer therapy that could directly target VEGFR2‐expressing tumor cells in addition to its anti‐angiogenic action., Affinity‐maturated anti‐vascular endothelial growth factor receptor 2 (VEGFR2) therapeutic Ab has strong clinical potential for cancer treatment due to its simultaneous inhibition of tumor angiogenesis in vascular endothelial cells and tumorigenesis in VEGFR2‐expressing tumor cells.

Hybrid molecules are composed of two pharmacophores with different biological activities. Here, we conjugated phthalazine moieties (antiangiogenetic pharmacophore) and bis(hydroxymethyl)pyrrole moieties (DNA cross-linking agent) to form a series of bis(hydroxymethyl)pyrrolo[2,1- a]phthalazine hybrids. These conjugates were cytotoxic to a variety of cancer cell lines by inducing DNA damage, arresting cell cycle progression at the G2/M phase, triggering apoptosis, and inhibiting vascular endothelial growth factor receptor 2 (VEGFR-2) in endothelial cells. Among them, compound 29d encapsulated in a liposomal formulation (e.g., 29dL) significantly suppressed the growth of small-cell lung cancer cell (H526) xenografts in mice. Based on immunohistochemical staining, the tumor xenografts in mice treated with 29dL showed time-dependent decreases in the intensity of CD31, a marker of blood vessels, whereas the intensity of γ-H2AX, a marker of DNA damage, increased. The present data revealed that the conjugation of antiangiogenic and DNA-damaging agents can generate potential hybrid agents for cancer treatment.

The viral E proteins of dengue virus (DENV) and Zika virus (ZIKV) are the major viral proteins involved in receptor binding and fusion, and for the induction of protective antibodies against viral infections. DIII of the E proteins is an independent domain and stretches out on the virion surface that can elicit type-specific neutralizing antibodies. For recombinant DIII vaccine development, prime-boost immunizations can provide an advantage of eliciting more type-specific neutralizing antibodies by recalling DIII antigens after DIII booster to improve protection. Methods: The DIII of the E genes of DENV and ZIKV were fused with bacterial fliC gene for the expression of flagellin-DIII (FliC-DIII) fusion proteins. Prime-boost immunization strategies by the second-dose booster of four DENV serotype or ZIKV FliC-DIII fusion proteins were used to investigate the induction of neutralizing antibodies and protection against viral infections. Cross-reactive non-neutralizing antibodies in each group of antisera were also examined using in vitro antibody-dependent enhancement (ADE) assay. A series of glycan-masking E antigens were finally constructed for prime-boost immunizations to abolish the elicitation of cross-reactive non-neutralizing antibodies for ADE activity. Results: We showed that inclusion of a bivalent live-attenuated vaccine with a FliC-DIII booster is superior in eliciting neutralization titers and protection in vivo against all four-serotype DENVs. We also demonstrated that recombinant adenovirus vectors encoding four-serotype DENV prMEs with a FliC-DIII prime-boost scheme is capable of eliciting good antibody responses. In contract, recombinant adenovirus vector of ZIKV prME gene priming, followed by ZIKV FliC-DIII booster did not improve vaccine efficacy. The glycan-masking mutation on the ZIKV E protein ij loop (E-248NHT), but not on DENV2 E protein ij loop (E-242NHT), resulted in abolishing the elicitation of cross-reactive antibodies for DENV and ZIKV infection enhancements. Conclusions: Our findings can provide useful information for designing novel immunogens and vaccination strategies in an attempt to develop a safe and efficacious DENV or ZIKV vaccine.

Development of effective therapeutics for mitigating the COVID-19 pandemic is a pressing global need. Neutralizing antibodies are known to be effective antivirals, as they can be rapidly deployed to prevent disease progression and can accelerate patient recovery without the need for fully developed host immunity. Here, we report the generation and characterization of a series of chimeric antibodies against the receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Some of these antibodies exhibit exceptionally potent neutralization activities in vitro and in vivo, and the most potent of our antibodies target three distinct non-overlapping epitopes within the RBD. Cryo-electron microscopy analyses of two highly potent antibodies in complex with the SARS-CoV-2 spike protein suggested they may be particularly useful when combined in a cocktail therapy. The efficacy of this antibody cocktail was confirmed in SARS-CoV-2-infected mouse and hamster models as prophylactic and post-infection treatments. With the emergence of more contagious variants of SARS-CoV-2, cocktail antibody therapies hold great promise to control disease and prevent drug resistance., Author summary Effective approaches to mitigate the COVID-19 pandemic are a pressing global need. One promising strategy is to combine neutralizing antibodies that can reduce viral load to prevent disease progression and accelerate patient recovery. However, the current supply of therapeutic antibodies for COVID-19 is insufficient to fill the enormous demand, and escape mutants may compromise the utility of existing drugs. Thus, there is an urgent worldwide need to develop highly potent neutralizing antibody cocktails. We generated a series of chimeric antibodies against the receptor-binding domain (RBD) of SARS-CoV-2 spike protein, which potently neutralize authentic SARS-CoV-2 infection according to the plaque reduction neutralization test (PRNT) and pseudovirus-based inhibition assay. These antibodies can be classified into three distinct groups based on their targets within the receptor-binding motif. Cryo-electron microscopy structural analyses of two representative receptor-binding domain-chimeric antibodies in complex with the SARS-CoV-2 spike protein further revealed two sets of non-overlapping epitopes, suggesting the potential for their combination in a therapeutic antibody cocktail. The prophylactic and therapeutic effects of these antibodies and their combination were demonstrated in SARS-CoV-2-infected mouse and hamster models. Thus, our potent neutralizing antibody cocktail has strong potential for development as an effective therapeutic drug to prevent and treat SARS-CoV-2 infection.

Although epithelial cell adhesion molecule (EpCAM) has previously been shown to promote tumor progression, the underlying mechanisms remain largely unknown. Here, we report that the EGF-like domain I within the extracellular domain of EpCAM (EpEX) binds EGFR, activating both AKT and MAPK signaling to inhibit forkhead transcription factor O3a (FOXO3a) function and stabilize PD-L1 protein, respectively. Treatment with the EpCAM neutralizing antibody, EpAb2-6, inhibited AKT and FOXO3a phosphorylation, increased FOXO3a nuclear translocation, and upregulated high temperature requirement A2 (HtrA2) expression to promote apoptosis while decreasing PD-L1 protein levels to enhance the cytotoxic activity of CD8+ T cells. In vivo, EpAb2-6 markedly extended survival in mouse metastasis and orthotopic models of human colorectal cancer. The combination of EpAb2-6 with atezolizumab, an anti-PD-L1 antibody, almost completely eliminated tumors. Moreover, the number of CD8+ T cells in combination-treated tumors was increased compared with atezolizumab alone. Our findings suggest a new combination strategy for cancer immunotherapy in patients with EpCAM-expressing tumors. Significance: This study shows that treatment with an EpCAM neutralizing antibody promotes apoptosis while decreasing PD-L1 protein to enhance cytotoxic activity of CD8+ T cells.

Epithelial cell adhesion molecule (EpCAM) is highly expressed in colon cancers, but its role in cancer progression remains to be elucidated. In this work, we found that the extracellular domain of EpCAM (EpEX) activated EGFR and downstream ERK1/2 signaling to promote colon cancer cell migration and proliferation, as well as tumor growth. Mechanistically, we discovered that EpEX-EGFR-ERK1/2 signaling positively regulated intramembrane proteolysis (RIP) of EpCAM and shedding of the intracellular domain (EpICD). Treatment with an EGFR inhibitor ablated the EpEX-induced phosphorylation of ERK1/2 and AKT. Additionally, treatment with inhibitors of either EGFR or MEK decreased EpEX-induced EpICD shedding and further revealed that EpICD is necessary for nuclear accumulation of β-catenin and the induction of HIF1α target gene expression in vitro and in vivo. Moreover, an anti-EpCAM neutralizing monoclonal antibody, EpAb2-6, inhibited the nuclear translocation of EpICD and β-catenin and induced apoptosis in colon cancer cells. Importantly, analysis of colorectal cancer tissues showed that nuclear accumulation of EpICD was highly correlated with metastasis and poor prognosis, suggesting that it may play an important functional role in cancer progression. Thus, we provide novel insights into the mechanisms and functions of EpEX-mediated signaling, which may be considered as a promising target for the treatment of colon cancer.

Background and objectives Squamous cell carcinoma of head and neck (SCCHN) is the fifth most prevalent cancer worldwide. Because the anatomical complexity of this region, complete surgical resection is often not achievable and conventional chemotherapy would aid locoregional control and mitigate distant metastasis. Nonetheless, the nonspecific cytotoxicity and short in vivo half-life of conventional chemotherapeutic drugs limit their effects. Given the high frequency of overexpression of wild type epidermal growth factor receptor (EGFR), we exploit EGFR as a homing beacon for drug delivery system with cytotoxic payloads. Materials and methods We generated fully human anti-EGFR single chain variable fragment (scFv)-conjugated immunoliposomes (IL) containing doxorubicin and vinorelbine and tested their anti-neoplastic efficacy in vitro and in vivo. Result Our IL enhanced endocytosis and significantly reduced the half maximal inhibitory concentrations of the therapeutic payloads when compared to non-targeting liposomal counterparts in various cell lines of SCCHN. Furthermore, median survival time was significantly prolonged in subcutaneous and orthotopic SCCHN xenograft murine models treated with our IL formulations than those treated with non-targeting counterparts (94 days versus 60 days and 72 days versus 56 days, respectively) without evident increased systemic toxicity. Conclusion The therapeutic index of the chemotherapeutic payloads was augmented by our EFGR-targeting IL formulation and they are warranted for further development and preclinical trial.

In patients who survive myocardial infarction, many go on to develop congestive heart failure (CHF). Despite ongoing efforts to develop new approaches for postinfarction therapy, there are still no effective therapeutic options available to CHF patients. Currently, the delivery of cardioprotective drugs relies entirely on passive uptake via the enhanced permeability and retention (EPR) effect which occurs in proximity to the infarction site. However, in ischemic disease, unlike in cancer, the EPR effect only exists for a short duration postinfarction and thus insufficient for meaningful cardioprotection. Splenic monocytes are recruited to the heart in large numbers postinfarction, and are known to interact with platelets during circulation. Therefore, the strategy is to exploit this interaction by developing platelet-like proteoliposomes (PLPs), biomimicking platelet interactions with circulating monocytes. PLPs show strong binding affinity for monocytes but not for endothelial cells in vitro, mimicking normal platelet activity. Furthermore, intravital multiphoton imaging shows that comparing to plain liposomes, PLPs do not aggregate on uninjured endothelium but do accumulate at the injury site 72 h postinfarction. Importantly, PLPs enhance the targeting of anti-inflammatory drug, cobalt protoporphyrin, to the heart in an EPR-independent manner, which result in better therapeutic outcome.

The objective of this study was to evaluate radiolabeled DOTA-SP90 as a radiotracer for breast cancer. The in vitro competition assay showed that radiolabeled DOTA-SP90 had significant binding affinity to BT-483 cancer cells. Biodistribution, nanoSPECT/CT and nanoPET/CT imaging results indicated that radiolabeled DOTA-SP90 can accumulate in tumors. In addition, radiolabeled DOTA-SP90 peptides can also detect metastatic tumors. Therefore, radiolabeled SP90 peptide may provide the potential capability as diagnostic agent for breast cancer patients.

Paclitaxel (PTX) exhibits potent antineoplastic activity against various human malignancies; however, clinical application must overcome the inherent hydrophobicity of this molecule. The commercialized Taxol formulation utilizes Cremophor EL (CrEL)/ethanol as a solvent to stabilize and dispense PTX in an aqueous solution. However, adverse CrEL-induced hypersensitivity reactions have been reported in ~30% of recipients, and 40% of patients receiving premedication may also experience this adverse effect. Therefore, the development of a CrEL-free delivery system is crucial, in order to fully exploit the therapeutic efficacy of PTX. In the present study, a novel liposomal PTX (lipo-PTX) formulation was optimized with regards to encapsulation rate and long-term stability, arriving at a molar constituent ratio of soybean phosp hatidylcholine:cholesterol:N-(carbonyl-methoxy-poly-ethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, sodium salt:PTX at 95:2:1:2. Comparable doses of lipo-PTX and Taxol were bioequivalent in terms of therapeutic efficacy in xenograft tumor models. However, the systemic side effects, including hematopoietic toxicity, acute hypersensitivity reactions and cardiac irregularities, were significantly reduced in lipo-PTX-treated mice compared with those infused with reference formulations of PTX. In conclusion, the present study reported that lipo-PTX exhibited a higher therapeutic index than clinical PTX formulations.

Dengue virus (DENV) infection is the most prevalent mosquito-borne viral infection of which there is no licensed therapeutic drug available. Previous studies have shown that minocycline, an antibiotic, can inhibit DENV infection in vitro. However, the mechanism is not fully understood. It is known that macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine, is involved in dengue disease development; MIF can induce autophagy, and autophagy can facilitate DENV replication. Therefore, we tested the hypothesis that MIF-induced autophagy is involved in minocycline treatment against DENV infection. We first showed that DENV infection induced MIF secretion and autophagy flux in HuH-7 cells. Suppression of endogenous MIF by short hairpin RNA (shRNA) and inhibition of MIF by its inhibitors attenuated DENV replication and autophagy formation. In addition, minocycline treatment suppressed DENV-induced MIF secretion and autophagy in vitro. Finally, we demonstrated that minocycline treatment attenuated viral load, MIF secretion, autophagy and increase survival in DENV-infected mice. These results suggest that inhibition of MIF-induced autophagy by minocycline might represent an alternative therapeutic approach against DENV infection.

Dengue virus (DENV) infection is currently at pandemic levels, with populations in tropical and subtropical regions at greatest risk of infection. Early diagnosis and management remain the cornerstone for good clinical outcomes, thus efficient and accurate diagnostic technology in the early stage of the disease is urgently needed. Serotype-specific monoclonal antibodies (mAbs) against the DENV1 nonstructural protein 1 (NS1), DA12-4, DA13-2, and DA15-3, which were recently generated using the hybridoma technique, are suitable for use in diagnostic platforms. Immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA) and Western blot analysis further confirmed the serotype specificity of these three monoclonal antibodies. The ELISA-based diagnostic platform was established using the combination of two highly sensitive mAbs (DA15-3 and DB20-6). The same combination was also used for the flow cytometry-based diagnostic platform. We report here the detection limits of flow cytometry-based and ELISA-based diagnostic platforms using these mAbs to be 0.1 and 1 ng/mL, respectively. The collected clinical patient serum samples were also assayed by these two serotyping diagnostic platforms. The sensitivity and specificity for detecting NS1 protein of DENV1 are 90% and 96%, respectively. The accuracy of our platform for testing clinical samples is more advanced than that of the two commercial NS1 diagnostic platforms. In conclusion, our platforms are suitable for the early detection of NS1 protein in DENV1 infected patients.

Nanocarriers, such as liposomes, have the potential to increase the payload of chemotherapeutic drugs while decreasing toxicity to non-target tissues; such advantageous properties can be further enhanced through surface conjugation of nanocarriers with targeting moieties. We previously reported that SP94 peptides, identified by phage display, exhibited higher binding affinity to human hepatocellular carcinoma (HCC) than to hepatocytes and other normal cells. Here, we confirm the tumor-targeting properties of SP94 peptide by near-infrared fluorescence imaging. Non-targeted PEGylated liposomal doxorubicin (LD) and SP94‑conjugated PEGylated liposomal doxorubicin (SP94‑LD) were compared by assessing pharmacokinetics, tissue distribution, and antitumor efficacy in xenograft-bearing mice, in order to investigate the effectiveness of SP94‑mediated targeting for cancer therapy. SP94‑LD demonstrated a significant increase in drug accumulation in tumors, while its plasma residence time was the same as its non-targeted equivalent. Consistent with this result, conjugation of targeting peptide SP94 enhances the therapeutic efficacy of liposomal doxorubicin in mouse models with hepatocellular carcinoma xenografts. Furthermore, combination targeted therapy exhibited a significant enhancement against orthotopic tumor growth, and markedly extended the survival of mice compared with all other treatments. Our study shows that SP94‑mediated targeting enhances antitumor efficacy by improving tumor pharmacokinetics and tissue distribution, allowing large amounts of antitumor drugs to accumulate in tumors.

Glutathione S-transferase mu 3 (GSTM3) is an enzyme involving in the detoxification of electrophilic compounds by conjugation with glutathione. Higher GSTM3 mRNA levels were reported in patients with ERα-positive breast cancer who received only tamoxifen therapy after surgery. Thus, this study aimed to clarify the oncogenic characteristics of GSTM3 in breast cancer and the mechanism of tamoxifen resistance. GSTM3 expression in human breast tumour tissues (n = 227) was analysed by RT-PCR and quantitative PCR. Western blot, promoter activity assays, and chromatin immunoprecipitation (ChIP) assays were used to investigate the mechanism of GSTM3 gene regulation. Hydrogen peroxide (H2O2)-induced cytotoxicity in breast cancer cells was detected by MTT assays and flow cytometry. The oncogenic characteristics of GSTM3 in MCF-7 cells were examined by siRNA knockdown in soft agar assays and a xenograft animal model. GSTM3 mRNA was highly expressed in ER- and HER2-positive breast cancers. Moreover, patients who received adjuvant Herceptin had increased GSTM3 mRNA levels in tumour tissue. Oestrogen-activated GSTM3 gene expression through ERα-mediated recruitment of SP1, EP300, and AP-1 complexes. GSTM3-silenced MCF-7 cells were more sensitive to H2O2, with significantly inhibited proliferation and colony formation abilities. Tamoxifen-resistant (Tam-R) cells lacking GSTM3 showed enhanced sensitivity to H2O2, but this result was contrary to that obtained after short-term tamoxifen exposure. The animal model suggested that GSTM3 silencing might suppress the tumourigenic ability of MCF-7 cells and increase tumour cell apoptosis. ROS production is one mechanism by which cancer drugs kill tumour cells, and according to our evidence, GSTM3 may play an important role in preventing breast cancer treatment-induced cellular cytotoxicity.

The anti-cancer efficacy of TNF-related apoptosis-inducing ligand (TRAIL)-based therapy is limited due to systemic toxicity, poor bioavailability, and the development of TRAIL-resistance. We developed a tumor-targeted LCPP (Lipid/Calcium/Phosphate/Protamine) nanoparticle (NP) to deliver TRAIL plasmid DNA (pDNA) into hepatocellular carcinoma (HCC) cells. TRAIL pDNA was encapsulated in a pH stimuli-responsive calcium phosphate (CaP) core, and protamine was added to facilitate nuclear delivery of pDNA. In addition, the intracellular release of Ca2+ from the CaP core overcame TRAIL resistance via calcium influx-dependent DR5 upregulation. TRAIL expression also attenuated fibrosis in the liver tissues surrounding HCCs by reverting activated hepatic stellate cells (HSCs) to a quiescent state or by directly inducing apoptosis in activated HSCs. Conclusion: TRAIL pDNA delivered by HCC-targeted LCPP NPs in combination with conventional sorafenib treatment attenuated HCC progression as well as liver fibrosis. Overall, our study presents an effective TRAIL-based cancer therapy that could be developed for clinical applications. This article is protected by copyright. All rights reserved.

Epithelial cell adhesion molecule (EpCAM) was reported to be cleaved into extracellular domain of EpCAM (EpEX) and intracellular domain of EpCAM (EpICD). We previously reported that EpCAM serves as a potent stem cell marker which is highly and selectively expressed by undifferentiated rather than differentiated hESC. However, the functional role of EpCAM remains elusive. Here, we found that EpEX and EpCAM enhance the efficiency of OSKM reprogramming. Interestingly, Oct4 or Klf4 alone, but not Sox2, can successfully reprogram fibroblasts into iPSCs with EpEX and EpCAM. Moreover, EpEX and EpCAM trigger reprogramming via activation of STAT3, which leads to the nuclear-translocation of HIF2α. This study reveals the importance of a novel EpEX/EpCAM-STAT3-HIF2α signal in the reprogramming process, and uncovers a new means of triggering reprogramming by delivery of soluble and transmembrane proteins.

Mutation of KRAS in non-small-cell lung cancer (NSCLC) shows a poor response to epidermal growth factor receptor (EGFR) inhibitors and chemotherapy. Currently, there are no direct anti-KRAS therapies available. Thus, new strategies have emerged for targeting KRAS downstream signaling. Panobinostat is a clinically available histone deacetylase inhibitor for treating myelomas and also shows potentiality in NSCLC. However, the therapeutic efficacy of panobinostat against gefitinib-resistant NSCLC is unclear. In this study, we demonstrated that panobinostat overcame resistance to gefitinib in KRAS-mutant/EGFR-wild-type NSCLC. Combined panobinostat and gefitinib synergistically reduced tumor growth in vitro and in vivo. Mechanistically, we identified that panobinostat-but not gefitinib-inhibited TAZ transcription, and the combination of panobinostat and gefitinib synergistically downregulated TAZ and TAZ downstream targets, including EGFR and EGFR ligand. Inhibition of TAZ by panobinostat or short hairpin RNA sensitized KRAS-mutant/EGFR-wild-type NSCLC to gefitinib through abrogating AKT/mammalian target of rapamycin (mTOR) signaling. Clinically, TAZ was positively correlated with EGFR signaling, and coexpression of TAZ/EGFR conferred a poorer prognosis in lung cancer patients. Our findings identify that targeting TAZ-mediated compensatory mechanism is a novel therapeutic approach to overcome gefitinib resistance in KRAS-mutant/EGFR-wild-type NSCLC.

Pancreatic adenocarcinoma is an aggressive disease with a high mortality rate. In this study, we have newly generated a monoclonal antibody (mAb), Pa65-2, which specifically binds to pancreatic cancer cells and tumor blood vessels. The target protein of Pa65-2 is identified as human clathrin heavy chain (CHC). In vitro and In vivo study showed that suppression of CHC either by shRNA or by Pa65-2 inhibited tumor growth and angiogenesis. One of the key functions of CHC was to bind with the hypoxia-inducing factor (HIF)-1α protein, increasing the stability of this protein and facilitating its nuclear translocation, thereby regulating the expression of VEGF. Taken together, our findings indicate that CHC plays a role in the processes of tumorigenesis and angiogenesis. Pa65-2 antibody or CHC shRNA can potentially be used for pancreatic cancer therapy.

Due to the development of drug resistance, the outcome for the majority of patients with acute myeloid leukemia (acute myelogenous leukemia; AML) remains poor. To prevent drug resistance and increase the therapeutic efficacy of treating AML, the development of new combinatory drug therapies is necessary. Sonic hedgehog (Shh) is expressed in AML biopsies and is essential for the drug resistance of cancer stem cells of AML. AML patients are frequently infected by bacteria and exposed to lipopolysaccharide (LPS). LPS itself, its derivatives, and its downstream effectors, such as tumor necrosis factor-α (TNF-α) and interferons (IFNs), have been shown to provoke anti-tumor effects. The application of a Shh inhibitor against AML cells in the presence of LPS/TNF-α/IFNs has not been investigated. We found that the Shh inhibitor cyclopamine in combination with LPS treatment synergistically induced massive cell apoptosis in THP-1 and U937 cells. The cytotoxic effects of this combined drug treatment were confirmed in 5 additional AML cell lines, in primary AML cells, and in an AML mouse model. Replacing cyclopamine with another Shh inhibitor, Sant-1, had the same effect. LPS could be substituted by TNF-α or IFNs to induce AML cell death in combination with cyclopamine. Our results suggest a potential strategy for the development of new therapies employing Shh antagonists in the presence of LPS/TNF-α/IFNs for the treatment of AML patients.

Building on the initial successful optimization of a novel series of tetraindoles, a second generation of the compounds with changes in the core phenyl ring was synthesized to improve anticancer properties. 17 new compounds with different rigidity, planarity, symmetry and degree of conjugation of their core structures to 5-hydroxyindole units were synthesized. All the compounds were fully characterized and tested against breast cancer cell line (MDA-MB-231). The results revealed that the core structure is required for activity and it should be aromatic, rigid, planar, symmetrical and conjugated for optimal activity. Compound 29, which has strong anticancer activity against various tumor-derived cell lines, including Mahlavu (hepatocellular), SK-HEP-1 (hepatic), HCT116 (colon), MIA PaCa-2 (pancreatic), H441 (lung papillary), A549 (lung), H460 (non-small cell lung) and CL1-5 (lung carcinoma) with IC50 values ranging from 0.19 to 3.50μM, was generated after series of successive optimizations. It was found to induce cell cycle arrest and apoptosis in vitro and inhibit tumor growth in the non-obese diabetic-severe combined immunodeficiency (NOD/SCID) mice bearing xenografted MIA PaCa-2 human pancreatic cancer.

The derivation of induced pluripotent stem cells (iPSCs) requires not only efficient reprogramming methods, but also reliable markers for identification and purification of iPSCs. Here, we demonstrate that surface markers, epithelial cells adhesion molecule (EpCAM) and epithelial cadherin (E-cadherin) can be used for efficient identification and/or isolation of reprogrammed mouse iPSCs. By viral transduction of Oct4, Sox2, Klf4 and n- or c-Myc into mouse embryonic fibroblasts, we observed that the conventional mouse embryonic stem cell (mESC) markers, alkaline phosphatase (AP) and stage-specific embryonic antigen 1 (SSEA1), were expressed in incompletely reprogrammed cells that did not express all the exogenous reprogramming factors or failed to acquire pluripotent status even though exogenous reprogramming factors were expressed. EpCAM and E-cadherin, however, remained inactivated in these cells. Expression of EpCAM and E-cadherin correlated with the activation of Nanog and endogenous Oct4, and was only seen in the successfully reprogrammed iPSCs. Furthermore, purification of EpCAM-expressing cells at late reprogramming stage by FACS enriched the Nanog-expressing cell population suggesting the feasibility of selecting successful reprogrammed mouse iPSCs by EpCAM expression. We have thus identified new surface markers that can efficiently identify successfully reprogrammed iPSCs and provide an effective means for iPSC isolation.

It is known that solid tumors recruit new blood vessels to support tumor growth, but the molecular diversity of receptors in tumor angiogenic vessels might also be used clinically to develop better targeted therapy. In vivo phage display was used to identify peptides that specifically target tumor blood vessels. Several novel peptides were identified as being able to recognize tumor vasculature but not normal blood vessels in severe combined immunodeficiency (SCID) mice bearing human tumors. These tumor-homing peptides also bound to blood vessels in surgical specimens of various human cancers. The peptide-linked liposomes containing fluorescent substance were capable of translocating across the plasma membrane through endocytosis. With the conjugation of peptides and liposomal doxorubicin, the targeted drug delivery systems enhanced the therapeutic efficacy of the chemotherapeutic agent against human cancer xenografts by decreasing tumor angiogenesis and increasing cancer cell apoptosis. Furthermore, the peptide-mediated targeting liposomes improved the pharmacokinetics and pharmacodynamics of the drug they delivered compared with nontargeting liposomes or free drugs. Our results indicate that the tumor-homing peptides can be used specifically target tumor vasculature and have the potential to improve the systemic treatment of patients with solid tumors.

Hepatocellular carcinoma is the fourth leading cause of cancer death worldwide. Novel treatment strategies derived from increased knowledge of molecular oncology are constantly being developed to cure this disease. Here, we used phage display to identify a novel peptide (SP94), which binds specifically to hepatocellular carcinoma cells. In vitro, the phage clone PC94 was shown to bind to hepatocellular carcinoma cell lines by ELISA and flow cytometry analysis. In vivo, PC94 homed specifically to tumor tissues but not to normal visceral organs in severe combined immunodeficient mice bearing human hepatocellular carcinoma xenografts. This homing ability could be competitively inhibited by synthetic peptide, SP94. Immunohistochemical staining confirmed that PC94 localized to tumor tissues and that it could not be detected in SP94-competed tumor tissues. In addition, PC94 recognized the tumor tissue but not nontumor tissue in surgical specimens from hepatocellular carcinoma patients, with a positive rate of 61.3% (19 of 31). With the conjugation of SP94 and liposomal doxorubicin, the targeted drug delivery system enhanced the therapeutic efficacy against hepatocellular carcinoma xenografts through enhanced tumor apoptosis and decreased tumor angiogenesis. Our results indicate that SP94 has the potential to improve the systemic treatment of patients with advanced hepatocellular carcinoma. [Mol Cancer Ther 2008;7(3):579–89]

Dengue virus (DEN), the pathogen behind dengue hemorrhagic fever, remains a public health problem in Asia and South America. In this study, monoclonal antibodies (MAbs) against DEN serotype 1 (DEN-1) were generated by fusing NSI/1-Ag4-1 mouse myeloma cells with lymphocytes from BALB/c mice immunized with DEN-1. Twelve MAbs were found to react specifically to the DENs by enzyme-linked immunosorbent assay, immunofluorescence analysis, and immunoblotting analysis. Five MAbs, namely, DA4-7, DA6-7, DA9-5, DA10-2, and DA11-13, were found to react with envelope proteins of DEN-1. Two serotype-specific MAbs of DEN-1, DA6-7 and DA11-13, were further shown to neutralize DEN-1 infection by a plaque reduction neutralization test. The neutralizing epitopes of these MAbs were further identified from a random peptide library displayed on phage. Immunopositive phage clones reacted specifically with these MAbs and did not react with normal mouse serum. Epitope-based peptide antigens were proved able to detect antibodies in serum samples collected from DEN-1-infected patients but not in those taken from DEN-2-infected patients or healthy controls. We believe that these MAbs and neutralizing epitopes will provide information that will lead to the development of DEN-1 serotype-specific diagnostic reagents and vaccines.

The four serotypes of dengue virus (DENV1-4) pose a serious threat to global health. Cross-reactive and non-neutralizing antibodies enhance viral infection, thereby exacerbating the disease via antibody-dependent enhancement (ADE). Studying the epitopes targeted by these enhancing antibodies would improve the immune responses against DENV infection. In order to investigate the roles of antibodies in the pathogenesis of dengue, we generated a panel of 16 new monoclonal antibodies (mAbs) against DENV4. Using plaque reduction neutralization test (PRNT), we examined the neutralizing activity of these mAbs. Furthermore, we used the in vitro and in vivo ADE assay to evaluate the enhancement of DENV infection by mAbs. The results indicate that the cross-reactive and poorly neutralizing mAbs, DD11-4 and DD18-5, strongly enhance DENV1-4 infection of K562 cells and increase mortality in AG129 mice. The epitope residues of these enhancing mAbs were identified using virus-like particle (VLP) mutants. W212 and E26 are the epitope residues of DD11-4 and DD18-5, respectively. In conclusion, we generated and characterized 16 new mAbs against DENV4. DD11-4 and D18-5 possessed non-neutralizing activities and enhanced viral infection. Moreover, we identified the epitope residues of enhancing mAbs on envelope protein. These results may provide useful information for development of safe dengue vaccine.

To understand whether the p53-regulated mdm2 gene expression was altered by the Epstein-Barr virus (EBV) in nasopharyngeal carcinoma (NPC), the NPC-TW01 cell line was infected by EBV through IgA receptor-mediated endocytosis. The mdm2 gene was expressed only in a small fraction of the NPC cell population and could be enhanced in the EBV-infected (EBV+) cells. In the animals bearing EBV+ and EBV- NPC xenografts, the MDM2+ cells only appeared in clusters in both EBV+ and EBV- tumors with stronger expression in EBV+ cells. Cotransfection of pmdm2-Luc plus pSV40-p53 plus pCMV-LMP1 in the NPC-TW06 line that had p53 heterozygous point mutation showed stronger mdm2 promoter activity than cells cotransfected with pmdm2-Luc plus pSV40-p53, but no mdm2 promoter activity was seen in cells cotransfected with pmdm2-Luc plus pCMV-LMP1. Only the EBV-LMP1 but not the EBV-LMP2A gene could enhance p53 to upregulated mdm2 expression. Tumor cells in NPC biopsy specimens revealed similar mdm2 expression as in the animal model. It is concluded that although EBV can indirectly enhance mdm2 gene expression in tumor cells that express this gene, it cannot turn on or directly regulate mdm2 expression in cells that do not express this gene. In other words, EBV plays a role as an enhancer in NPC tumorigenesis.

In this study, a serotype-specific monoclonal antibody (mAb), D216-1 (Ab4), against dengue virus type 2 (DEN-2) was generated. The specificity of Ab4, which recognized DEN-2 non-structural protein 1, was determined by ELISA, immunofluorescence and immunoblotting analyses. The serotype-specific B-cell epitope of Ab4 was identified further from a random phage-displayed peptide library; selected phage clones reacted specifically with Ab4 and did not react with other mAbs. Immunopositive phage clones displayed a consensus motif, His–Arg/Lys–Leu/Ile, and a synthetic peptide corresponding to the phage-displayed peptide bound specifically to Ab4. The His and Arg residues in this epitope were found to be crucial for peptide binding to Ab4 and binding activity decreased dramatically when these residues were changed to Leu. The epitope-based synthetic peptide not only identified serum samples from DEN-2-immunized mice and rabbits by ELISA but also differentiated clearly between serum samples from DEN-2- and Japanese encephalitis virus-immunized mice. This mAb and its epitope-based peptide antigen will be useful for serologic diagnosis of DEN-2 infection. Furthermore, DEN-2 epitope identification makes it feasible to dissect antibody responses to DEN and to address the role of antibodies in the pathogenesis of primary and secondary DEN-2 infections.

Studies of the pathogenesis of dengue hemorrhagic fever (DHF), a potentially life-threatening disease, have revealed the importance of initial high levels of virus replication. However, the possible involvement of virus during the transition from fever to defervescence, a critical stage in determining the severity of disease, has not been appreciated. Using quantitative reverse transcription-polymerase chain reaction, we examined the levels of plasma dengue viral load during both fever and defervescence periods in patients from a DEN-3 outbreak in southern Taiwan in 1998. Higher levels of plasma dengue viral RNA were found in DHF patients than in DF patients. During defervescence, while the level of plasma dengue viral RNA was undetectable in most DF patients, it remains high in all DHF patients. Using a modified immunoprecipitation assay, we demonstrated for the first time that the plasma dengue viruses persisting during defervescence were in the immune complexes for most DHF patients. These findings suggest that continued active viral replication or delay in the clearance of viremia contributes to the pathogenesis of DHF. Moreover, high levels of plasma dengue viral RNA during defervescence may serve as a disease marker for DHF.

We have generated a novel monoclonal antibody (mAb), OCAb9-1, which specifically binds to various types of cancer cell lines, but not to normal cells. According to the results of immunoaffinity chromatography, LC-MS/MS analysis, co-immunoprecipitation, and RNA interference studies, the target protein of OCAb9-1 is the epithelial cell adhesion molecule (EpCAM). EpCAM is a type I transmembrane glycoprotein which is highly expressed in epithelial-transformed neoplasia and tumor-initiating cells (TICs). However, regulation of EpCAM gene expression in tumors and its role in tumorigenesis are not fully understood. In the present study, we show that EpCAM expression is elevated in several cancer cell lines and tumor tissues. Loss-of-function experiments were performed to demonstrate that EpCAM negatively regulates expression of p53 and p21, and promotes tumor cell growth, colony formation, migration and invasion. The median overall survival of tumor-bearing mice treated with OCAb9-1 was significantly higher than that of PBS-treated mice. Moreover, we report that the interplay between SUZ12 and JMJD3 results in dynamic regulation of lysine 27 trimethylation of histone 3 (H3K27me3). Taken together, our findings suggest that the anti-EpCAM mAb may be suitable for use in cancer diagnosis, prognosis, imaging and therapy. Furthermore, EpCAM overexpression in cancer cells is strongly associated with tumor progression, and may be regulated by epigenetic mechanisms.

Clostridium botulinumneurotoxin type A (BTx-A) is known to inhibit the release of acetylcholine at the neuromuscular junctions and synapses and to cause neuroparalysis and death. In this study, we have identified two monoclonal antibodies, BT57-1 and BT150-3, which protect ICR mice against lethal doses of BTx-A challenge. The neutralizing activities for BT57-1 and BT150-3 were 103and 104times the 50% lethal dose, respectively. Using immunoblotting analysis, BT57-1 was recognized as a light chain and BT150-3 was recognized as a heavy chain of BTx-A. Also, applying the phage display method, we investigated the antibodies' neutralizing B-cell epitopes. These immunopositive phage clones displayed consensus motifs, Asp-Pro-Leu for BT57-1 and Cys-X-Asp-Cys for BT150. The synthetic peptide P4M (KGTFDPLQEPRT) corresponded to the phage-displayed peptide selected by BT57-1 and was able to bind the antibodies specifically. This peptide was also shown by competitive inhibition assay to be able to inhibit phage clone binding to BT57-1. Aspartic acid (D5) in P4M was crucial to the binding of P4M to BT57-1, since its binding activity dramatically decreased when it was changed to lysine (K5). Finally, immunizing mice with the selected phage clones elicited a specific humoral response against BTx-A. These results suggest that phage-displayed random-peptide libraries are useful in identifying the neutralizing epitopes of monoclonal antibodies. In the future, the identification of the neutralizing epitopes of BTx-A may provide important information for the identification of the BTx-A receptor and the design of a BTx-A vaccine.

A T60M mutation in the thiazide-sensitive sodium chloride cotransporter (NCC) is common in patients with Gitelman's syndrome (GS). This mutation prevents Ste20-related proline and alanine-rich kinase (SPAK)/oxidative stress responsive kinase-1 (OSR1)-mediated phosphorylation of NCC and alters NCC transporter activity in vitro. Here, we examined the physiologic effects of NCC phosphorylation in vivo using a novel Ncc T58M (human T60M) knock-in mouse model. Ncc(T58M/T58M) mice exhibited typical features of GS with a blunted response to thiazide diuretics. Despite expressing normal levels of Ncc mRNA, these mice had lower levels of total Ncc and p-Ncc protein that did not change with a low-salt diet that increased p-Spak. In contrast to wild-type Ncc, which localized to the apical membrane of distal convoluted tubule cells, T58M Ncc localized primarily to the cytosolic region and caused an increase in late distal convoluted tubule volume. In MDCK cells, exogenous expression of phosphorylation-defective NCC mutants reduced total protein expression levels and membrane stability. Furthermore, our analysis found diminished total urine NCC excretion in a cohort of GS patients with homozygous NCC T60M mutations. When Wnk4(D561A/+) mice, a model of pseudohypoaldosteronism type II expressing an activated Spak/Osr1-Ncc, were crossed with Ncc(T58M/T58M) mice, total Ncc and p-Ncc protein levels decreased and the GS phenotype persisted over the hypertensive phenotype. Overall, these data suggest that SPAK-mediated phosphorylation of NCC at T60 regulates NCC stability and function, and defective phosphorylation at this residue corrects the phenotype of pseudohypoaldosteronism type II.

Targeted delivery of drugs to tumors represents a significant advance in cancer diagnosis and therapy. Therefore, development of novel tumor-specific ligands or pharmaceutical nanocarriers is highly desirable. In this study, we utilized phage display to identify a new targeting peptide, SP90, which specifically binds to breast cancer cells, and recognizes tumor tissues from breast cancer patients. We used confocal and electron microscopy to reveal that conjugation of SP90 with liposomes enables efficient delivery of drugs into cancer cells through endocytosis. Furthermore, in vivo fluorescent imaging demonstrated that SP90-conjugated quantum dots possess tumor-targeting properties. In tumor xenograft and orthotopic models, SP90-conjugated liposomal doxorubicin was found to improve the therapeutic index of the chemotherapeutic drug by selectively increasing its accumulation in tumors. We conclude that the targeting peptide SP90 has significant potential in improving the clinical benefits of chemotherapy in the treatment and the diagnosis of breast cancer.

Nucleolin (C23, NCL) mRNA was up-regulated in nasopharyngeal carcinoma (NPC) cells compared to that of normal nasomucosal (NNM) cells using a cDNA microarray approach. The level of nucleolin protein was also up-regulated in 13 NPC cell lines, 30 biopsy specimens and nine other cancer cell lines compared to five NNM cells or normal stromal cells, which were analyzed using immunoblotting or immunohistochemistry. We transfected nucleolin antisense oligodeoxynucleotides (phosphorothioate-modified oligodeoxynucleotides; S-ODNs) into NPC-TW01 cells to knockdown nucleolin expression to evaluate the function of nucleolin in cancer cells. Nucleolin knockdown induced NPC cells but not NNM cells to undergo apoptosis. Furthermore, treatment of NPC-TW01 xenograft tumors with nucleolin antisense oligodeoxynucleotides suppressed the growth of xenograft tumors without obvious side effects. Therefore, we suggest that nucleolin may be a potential cancer therapeutic target and that nucleolin antisense oligodeoxynucleotides may be used as a potential drug for therapy in NPC.

Aberrantly expressed c-Met, the receptor for hepatocyte growth factor (HGF), has been implicated in human lung cancer as well as malignancy, metastasis and drug-resistance in other human cancers. Thus, this molecule could be a potential target for antibody-based cancer therapy. Targeting delivery of compound to tumor represented benefit for cancer detection and therapy. In this study, we utilized phage display to identify human single chain variable fragment (scFv) antibodies that specifically bound to c-Met protein. The anti-c-Met scFvs selectively bound to and internalized in several lung cancer cell lines expressing c-Met. Conjugation of anti-c-Met scFv with PEGylated liposomes enabled the efficient delivery of doxorubicin into cancer cells where it exerted cytotoxic activity by inducing apoptosis pathway. In addition, in vivo fluorescent imaging by scFv-conjugated quantum dots showed higher tumor uptake and increased tumor-normal tissue ratios. In a tumor xenograft model, anti-c-Met immunoliposome was found to selectively increase tumor accumulation of a chemotherapeutic drug and enhance its antitumor activity. Taken together, our results suggest that anti-c-Met scFv-mediated drug delivery systems show great promise in tumor-targeted therapy and imaging.

Nasopharyngeal carcinoma (NPC) is prevalent in south-eastern Asia, particularly southern China, Singapore and Taiwan. The aim of this study was to identify the pivotal genes that may be altered during NPC progression. Using cDNA microarray analysis, we compared the expression of 18 genes between NPC and normal nasomucosal cells. qRT-PCR analysis found the expression of IBFBP-6 in NPC cell lines and immunolocalization of IGFBP-6 in NPC to be very weak. To explore the effects of IGFBP-6 on NPC tumour growth, we constructed inducible plasmids containing full-length IGFBP-6 cDNA (pBIG2i-IGFBP-6) and established pBIG2i-IGFBP-6-transfected NPC stable cell lines (NPC-TW01-pBIG2i-IGFBP-6). We then performed functional analysis of the IGFBP-6 in cell lines in vitro and in vivo. Over-expression of IGFBP-6 significantly suppressed the proliferation, invasion and metastatic activity of NPC cells and increased their apoptosis. We found the EGR-1, caspase-1 and TSP-1 genes to be markedly up-regulated when NPC-pBIG2i-IGFBP-6 was treated with doxycycline. Knocking down EGR-1 with EGR-1 siRNA resulted in a decrease in expression of caspase-1, TSP-1 and EGR-1 but not the expression of IGFBP-6. However, in knockdown cells the unchanged expression of IGFBP-6 did not inhibit the migration of NPC cells. Chromatin immunoprecipitation and luciferase reporter assay experiments showed that IGFBP-6 bound the EGR-1 promoter regions and activated EGR-1 promoter. We conclude that IGFBP-6 can regulate the progression of NPC by regulating the expression of EGR-1. These results suggest that IGFBP-6 could be used as a new target in NPC therapy.

Nasopharyngeal carcinoma (NPC) is one of the most common cancers among Chinese living in South China, Singapore, and Taiwan. At present, its etiological factors are not well defined. To identify which genetic alterations might be involved in NPC pathogenesis, we identified genes that were differentially expressed in NPC cell lines and normal nasomucosal cells using subtractive hybridization and microarray analysis. Most NPC cell lines and biopsy specimens were found to have higher expression levels of the gene encoding nucleolar and coiled-body phosphoprotein 1 (NOLC1) as compared with normal cells. Severe combined immunodeficiency mice bearing NPC xenografts derived from NOLC1-short hairpin-RNA-transfected animals were found to have 82% lower levels of tumor growth than control mice as well as marked tumor cell apoptosis. Measuring the expression levels of genes related to cell growth, apoptosis, and angiogenesis, we found that the MDM2 gene was down-regulated in the transfectants. Both co-transfection and chromatin immunoprecipitation experiments showed that tumor protein 53-regulated expression of the MDM2 gene requires co-activation of NOLC1. These findings suggest that NOLC1 plays a role in the regulation of tumorigenesis of NPC and demonstrate that both NOLC1 and tumor protein 53 work together synergistically to activate the MDM2 promoter in NPC cells.

Nasopharyngeal carcinoma (NPC) is prevalent in south-eastern Asia, and its tumourigenesis is rather complex. The purpose of this research was to identify the pivotal genes that may be altered during the early stage of NPC progression. Eleven genes were selected by comparative microarray analysis of NPC versus normal nasomucosal cells. The expression of SPARC (secreted protein, acidic, cysteine-rich) was statistically significantly down-regulated in NPC cells. In exploring the mechanism underlying the decreased transcription of SPARC in NPC cells, we found that the transcription factor SRY (sex-determining region Y)-box 5 (SOX-5) is up-regulated in NPC cells. RNA interference of SOX-5 by short hairpin RNA (shRNA) in NPC cells caused a dramatic increase in SPARC and chromosome immunoprecipitation assay showed that SOX-5 can bind directly to the SPARC promoter, suggesting that SOX-5 acts as a key transcriptional repressor of SPARC. We further demonstrated that shRNA knockdown of SOX-5 suppressed the proliferation of NPC cells, as well as their migratory ability, which was also observed when SPARC was over-expressed in NPC cells. Alternatively, blocking SPARC with an antagonistic antibody reversed the effects of SOX-5 knockdown. In 66 NPC patients, over-expression of SOX-5 in tumour cells correlated clinically with poor survival. Our study suggests that SOX-5 transcriptionally down-regulates SPARC expression and plays an important role in the regulation of NPC progression. SOX-5 is a potential tumour marker for poor NPC prognosis.

Antiangiogenesis therapies for the treatment of cancers hold the promise of high efficacy and low toxicity. In vivo phage display was used to identify peptides specifically targeting tumor blood vessels. The peptide SP5-52 recognized tumor neovasculature but not normal blood vessels in severe combined immunodeficiency mice bearing human tumors. Synthetic peptide was shown to inhibit the binding of PC5-52 phage particles to the tumor mass in the competitive inhibition assay. Several selected phage clones displayed the consensus motif, proline-serine-proline, and this motif was crucial for peptide binding to the tumor neovasculature. SP5-52 peptides also bound vascular endothelial growth factor–stimulated human umbilical vein endothelial cells and blood vessels of human lung cancer surgical specimens. Furthermore, this targeting phage was shown to home to tumor tissues from eight different types of human tumor xenografts following in vivo phage display experiments. An SP5-52 peptide-linked liposome carrying doxorubicin enhanced the therapeutic efficacy of the drug, markedly decreased tumor blood vessels, and resulted in higher survival rates of human lung and oral cancer–bearing xenograft mice. The current study indicates that ligand-targeted therapy offers improved therapeutic effects over conventional anticancer drug therapy, and that the peptide SP5-52 specifically targets tumor neovasculature and is a good candidate for targeted drug delivery to solid tumors. [Cancer Res 2007;67(22):10958–65]

Dengue virus (DENV), a global disease, is divided into four serotypes (DENV1-4). Cross-reactive and non-neutralizing antibodies against envelope (E) protein of DENV bind to the Fcγ receptors (FcγR) of cells, and thereby exacerbate viral infection by heterologous serotypes via antibody-dependent enhancement (ADE). Identification and modification of enhancing epitopes may mitigate enhancement of DENV infection. In this study, we characterized the cross-reactive DB21-6 and DB39-2 monoclonal antibodies (mAbs) against domain I-II of DENV; these antibodies poorly neutralized and potently enhanced DENV infection both in vitro and in vivo. In addition, two enhancing mAbs, DB21-6 and DB39-2, were observed to compete with sera antibodies from patients infected with dengue. The epitopes of these enhancing mAbs were identified using phage display, structural prediction, and mapping of virus-like particle (VLP) mutants. N8, R9, V12, and E13 are the reactive residues of DB21-6, while N8, R9, and E13 are the reactive residues of DB39-2. N8 substitution tends to maintain VLP secretion, and decreases the binding activity of DB21-6 and DB39-2. The immunized sera from N8 substitution (N8R) DNA vaccine exerted greater neutralizing and protective activity than wild-type (WT)-immunized sera, both in vitro and in vivo. Furthermore, treatment with N8R-immunized sera reduced the enhancement of mortality in AG129 mice. These results support identification and substitution of enhancing epitope as a novel strategy for developing safe dengue vaccines., Author Summary Dengue virus (DENV) infects 390 million humans annually, and is the cause of one of the most important arthropod-borne viral diseases in the world. Currently, there are no available licensed vaccines or antiviral drugs for dengue, so development of safe vaccine and effective therapy is urgently needed. Here, we identified two monoclonal antibodies, DB21-6 and DB39-2, which can enhance DENV1-4 infection and increase virus-induced mortality in AG129 mice. We found that serum samples from patients with severe dengue disease contain higher levels of antibodies against enhancing epitope. We proceeeded to identify enhancing epitope on E protein, and developed DNA vaccines by substitution. The substituted DNA vaccine with mutation at the enhancing epitope demonstrated augmented neutralizing activity against DENV2, and reduced enhancement of mortality as compared to wild type-immunized sera. Our results show that substitution of enhancing epitope can increase the immune response against viral infection, while reducing the potential for antibody-dependent enhancement (ADE). These novel findings may be useful for developing safe and efficacious vaccines against dengue.

Nasopharyngeal carcinoma (NPC) is a common cancer among Chinese living in southern China, Taiwan, and Singapore. The 5-year survival rate in the early stage of NPC has been reported as high as 90 to 95% with the use of radiotherapy, but in the advanced cases, even with the use of both chemotherapy and radiotherapy, the survival rate is still

A membrane invasion culture system was used to study the ability of EBV to enhance invasion and migration of nasopharyngeal carcinoma (NPC) cells. Semi-reverse transcriptase-PCR analysis of matrix proteinases and angiogenic factors from EBV-infected, or EBV-positive (EBV+), cells demonstrated different degrees of elevated gene expression. In our animal model, EBV+ tumors grew faster and larger than EBV-free, or EBV-negative (EBV-), tumors and also had clonal EBV terminal repeat sequences. Double-localization of EBV and certain host proteins in EBV+ tumors and biopsy specimens demonstrated that EBV up-regulates host genes only in cells that express those genes but not in cells that do not express them. Double-localization of EBV and host genes in NPC biopsy specimens all showed EBV- tumor cells expressing those host genes. Our data strongly suggest that EBV infection enhances progression of NPC tumor growth. They do not rule out a role for EBV infection in the induction and early promotion of NPC development. Unidentified factors may also enhance NPC tumor growth independent of the effects of EBV.

Lung cancer ranks among the most common malignancies, and is the leading cause of cancer-related mortality worldwide. Chemotherapy for lung cancer can be made more specific to tumor cells, and less toxic to normal tissues, through the use of ligand-mediated drug delivery systems. In this study, we investigated the targeting mechanism of the ligand-mediated drug delivery system using a peptide, SP5-2, which specifically binds to non-small cell lung cancer (NSCLC) cells. Conjugation of SP5-2 to liposomes enhanced the amount of drug delivered directly into NSCLC cells, through receptor-mediated endocytosis. Functional SP5-2 improved the therapeutic index of Lipo-Dox by enhancing therapeutic efficacy, reducing side effects, and increasing the survival rate of tumor-bearing mice in syngenic, metastatic and orthotopic animal models. Accumulation of SP5-2-conjugated liposomal doxorubicin (SP5-2-LD) in tumor tissues was 11.2-fold higher than that of free doxorubicin, and the area under the concentration-time curve (AUC0-72 hours) was increased 159.2-fold. Furthermore, the experiment of bioavailability was assessed to confirm that SP5-2 elevates the uptake of the liposomal drugs by the tumor cells in vivo. In conclusion, the use of SP5-2-conjugated liposomes enhances pharmacokinetic properties, improves efficacy and safety profiles, and allows for controlled biodistribution and drug release.