Browsing by Author "Chou, Min-Yuan"

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Antiviral Activity of a Llama-Derived Single-Domain Antibody against Enterovirus A71
(2020-05-01)
Huang, Peng-Nien
;
Wang, Hsiang-Ching
;
Hung, Hui-Chen
;
Tseng, Sung-Nien
;
Chang, Teng-Yuan
In the past few decades, enterovirus A71 (EVA71) has caused devastating outbreaks in the Asia-Pacific region, resulting in serious sequelae in infected young children. No preventive or therapeutic interventions are currently available for curing EVA71 infection, highlighting a great unmet medical need for this disease. Here, we showed that one novel single-domain antibody (sdAb), F1, isolated from an immunized llama, could alleviate EVA71 infection both in vitro and in vivo. We also confirmed that the sdAb clone F1 recognizes EVA71 through a novel conformational epitope comprising the highly conserved region of VP3 capsid protein by using competitive-binding and overlapping-peptide enzyme-linked immunosorbent assays (ELISAs). Because of the virion's icosahedral structure, we reasoned that adjacent epitopes must be clustered within molecular ranges that may be simultaneously bound by an engineered antibody with multiple valency. Therefore, two singledomain binding modules (F1) were fused to generate an sdAb-in-tandem design so that the capture of viral antigens could be further increased by valency effects. We showed that the tetravalent construct F1xF1-hFc, containing two sdAb-intandem on a fragment crystallizable (Fc) scaffold, exhibits more potent neutralization activity against EVA71 than does the bivalent sdAb F1-hFc by at least 5.8-fold. We also demonstrated that, using a human scavenger receptor class B member 2 (hSCARB2) transgenic mouse model, a half dose of the F1xF1-hFc provided better protection against EVA71 infection than did the F1-hFc. Thus, our study furnishes important insights into multivalent sdAb engineering against viral infection and provides a novel strategic deployment approach for preparedness of emerging infectious diseases such as EVA71.
Bispecific Antibody Conjugated Manganese-Based Magnetic Engineered Iron Oxide for Imaging of HER2/neu- and EGFR-Expressing Tumors
(2016-01-01)
Wu, Shou-Cheng
;
Chen, Jen
;
Wang, Hsiang-Ching
;
Chou, Min-Yuan
;
Chang, Teng-Yuan
The overexpression of HER2/neu and EGFR receptors plays important roles in tumorigenesis and tumor progression. Targeting these two receptors simultaneously can have a more widespread application in early diagnosis of cancers. In this study, a new multifunctional nanoparticles (MnMEIO-CyTE777-(Bis)-mPEG NPs) comprising a manganese-doped iron oxide nanoparticle core (MnMEIO), a silane-amino functionalized poly(ethylene glycol) copolymer shell, a near infrared fluorescence dye (CyTE777), and a covalently conjugated anti-HER2/neu and anti-EGFR receptors bispecific antibody (Bis) were successfully developed. In vitro T-2-weighted MR imaging studies in SKBR-3 and A431 tumor cells incubated with MnMEIO-CyTE777-(Bis)-mPEG NPs showed -94.8 +/- 3.8 and -84.1 +/- 2.8% negative contrast enhancement, respectively. Pharmacokinetics study showed that MnMEIO-CyTE777-(Bis)-mPEG NPs were eliminated from serum with the half-life of 21.3 mins. In vivo MR imaging showed that MnMEIO-CyTE777-(Bis)-mPEG NPs could specifically and effectively target to HER2/neu-and EGFR-expressing tumors in mice; the relative contrast enhancements were 11.8 (at 2 hrs post-injection) and 61.5 (at 24 hrs post-injection) fold higher in SKBR-3 tumors as compared to Colo-205 tumors. T2-weighted MR and optical imaging studies revealed that the new contrast agent (MnMEIO-CyTE777-(Bis)-mPEG NPs) could specifically and effectively target to HER2/neu-and/or EGFR-expressing tumors. Our results demonstrate that MnMEIO-CyTE777-(Bis)-mPEG NPs are able to recognize the tumors expressing both HER2/neu and/or EGFR, and may provide a novel molecular imaging tool for early diagnosis of cancers expressing HER2/neu and/or EGFR.
Silicon Quantum Dot Nanoparticles with Antifouling Coatings for Immunostaining on Live Cancer Cells
(2016-06-08)
Tu, Chang-Ching
;
Chen, Kuang-Po
;
Yang, Tsu-An
;
Chou, Min-Yuan
;
Lin, Lih Y.
Fluorescent silicon quantum dots (SiQDs) have shown a great potential as antiphotobleaching, nontoxic and biodegradable labels for various in vitro and in vivo applications. However, fabricating SiQDs with high water-solubility and high photoluminescence quantum yield (PLQY) remains a challenge. Furthermore, for targeted imaging, their surface chemistry has to be capable of conjugating to antibodies, as well as sufficiently antifouling. Herein, antibody-conjugated SiQD nanoparticles (SiQD-NPs) with antifouling coatings composed of bovine serum albumin (BSA) and polyethylene glycol (PEG) are demonstrated for immunostaining on live cancer cells. The monodisperse SiQD-NPs of diameter about 130 nm are synthesized by a novel top-down method, including electrochemical etching, photochemical hydrosilylation, high energy ball milling, and "selective-etching" in HNO3 and HF. Subsequently, the BSA and PEG are covalently grafted on to the SiQD-NP surface through presynthesized chemical linkers, resulting in a stable, hydrophilic, and antifouling organic capping layer with isothiocyanates as the terminal functional groups for facile conjugation to the antibodies. The in vitro cell viability assay reveals that the BSA-coated SiQD-NPs had exceptional biocompatibility, with minimal cytotoxicity at concentration up to 1600 mu g mL(-1). Under 365 nm excitation, the SiQD-NP colloid emits bright reddish photoluminescence with PLQY = 45-55% in organic solvent and 5-10% in aqueous buffer. Finally, through confocal fluorescent imaging and flow cytometry analysis, the anti-HER2 conjugated SiQD-NPs show obvious specific binding to the HER2-overexpressing SKOV3 cells and negligible nonspecific binding to the HER2-nonexpressing CHO cells. Under similar experimental conditions, the immunofluorescence results obtained with the SiQD-NPs are comparable to those using conventional fluorescein isothiocyanate (FITC).