Browsing by Author "Hsieh, Yuan-Chin"

Now showing 1 - 7 of 7

A regularly spaced and self-revealing protein ladder for anti-tag Western blot analysis
(2012-12-01)
Kao, Chien-Han
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Cheng, Chiu-Min
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Chuang, Kuo-Hsiang
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Chuang, Chih-Hung
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Tzou, Shey-Cherng
We designed a protein ladder (hereafter referred to as "Mega-tag") that contains 14 of the most commonly used epitope tags fused to molecular weight markers. The Mega-tag ladder can be simultaneously visualized when anti-tag antibodies are used to detect epitope-tagged recombinant proteins in Western blots. The logarithm of molecular weights and relative mobility of the Mega-tag protein ladder are highly correlated (R-2 = 0.997 +/- 0.00232), indicating that the dye-free Mega-tag protein ladder is accurate. It can also serve as a positive control for anti-epitope tag immunoblots. The Mega-tag protein ladder should provide a convenient and precise tool for Western blot analysis. (c) 2012 Elsevier Inc. All rights reserved.
Bispecific antibody (HER2 x mPEG) enhances anti-cancer effects by precise targeting and accumulation of mPEGylated liposomes
(2020-07-15)
Chen, I-Ju
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Cheng, Yi-An
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Ho, Kai-Wen
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Lin, Wen-Wei
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Cheng, Kai-Wen
Targeted antibodies and methoxy-PEGylated nanocarriers have gradually become a mainstream of cancer therapy. To increase the anti-cancer effects of targeted antibodies combined with mPEGylated liposomes (mPEG-liposomes), we describe a bispecific antibody in which an anti-methoxy-polyethylene glycol scFv (alpha mPEG scFv) was fused to the C-terminus of an anti-HER2 (alpha HER2) antibody to generate a HER2 x mPEG BsAb that retained the original efficacy of a targeted antibody while actively attracting mPEG-liposomes to accumulate at tumor sites. HER2 xmPEG BsAb can simultaneously bind to HER2high expressing MCF7/HER2 tumor cells and mPEG molecules on mPEG-liposomal doxorubicin (Lipo-Dox). Pre-incubation of HER2 x mPEG BsAb with cells increased the endocytosis of Lipo-DiD and enhanced the cytotoxicity of Lipo-Dox to MCF7/HER2 tumor cells. Furthermore, pre-treatment of HER2 x mPEG BsAb enhanced the tumor accumulation and retention of Lipo-DiR 2.2-fold in HER2-high expressing MCF7/HER2 tumors as compared to HER2-low expressing MCF7/neol tumors. Importantly, HER2 x mPEG BsAb plus Lipo-Dox significantly suppressed tumor growth as compared to control BsAb plus Lipo-Dox in MCF7/HER2 tumor-bearing mice. These results indicate that HER2 x mPEG BsAb can enhance tumor accumulation of mPEG-liposomes to improve the therapeutic efficacy of combination treatment. Anti-mPEG scFv can be fused to any kind of targeted antibody to generate BsAbs to actively attract mPEG-drugs and improve anti-cancer efficacy. Statement of Significance Antibody targeted therapy and PEGylated drugs have gradually become the mainstream of cancer therapy. To enhance the anti-cancer effects of targeted antibodies combined with PEGylated drugs is very important. To this aim, we fused an anti-PEG scFv to the C-terminal of HER2 targeted antibodies to generate a HER2xmPEG bispecific antibody (BsAb) to retain the original efficacy of targeted antibody whilst actively attract mPEG-liposomal drugs to accumulate at tumor sites. The present study demonstrates pretreatment of HER2xmPEG BsAb can enhance tumor accumulation of mPEG-liposomal drugs to improve the therapeutic efficacy of combination treatment. Anti-mPEG scFv can be fused to any kind of targeted antibody to generate BsAbs to actively attract mPEG-drugs and improve anti-cancer efficacy. (C) 2020 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
Development of a highly sensitive enzyme-linked immunosorbent assay (ELISA) through use of polyprotein G-expressing cell-based microplates
(2018-12-14)
Chen, Yi-Jou
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Chen, Michael
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Hsieh, Yuan-Chin
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Su, Yu-Cheng
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Wang, Chang-Hung
The sensitivity of traditional enzyme-linked immunosorbent assays (ELISAs) is limited by the low binding avidity and heterogeneous orientation of capture antibodies coated on polystyrene-based microplates. Here, we developed a highly sensitive ELISA strategy by fixing poly-protein G-expressing cells on microplates to improve the coating amount and displayed orientation of capture antibodies. One or eight repeated fragment crystallisable (Fc) binding domains of protein G are stably expressed on the surface of BALB/c 3T3 cells (termed 1pG cells or 8pG cells), which then act as highly antibody-trapping microparticles. The 8pG cells showed higher antibody-trapping ability than the 1pG cells did. The antibody-coating amount of the 8pG cell-based microplates was 1.5-23 times and 1.2-6.8 times higher than that of traditional polystyrene-based and commercial protein G-based microplates, respectively. The 8pG cell-based microplates were then applied to an anti-IFN-alpha sandwich ELISA and an anti-CTLA4 competitive ELISA, respectively, and dramatically enhanced their detection sensitivity. Importantly, direct coating unpurified capture antibody produced by mammalian cells did not impair the antigen-capturing function of 8pG cell-based microplates. The 8pG cell-based microplates exhibited a significant improvement in antibody-coating amount and preserved the homogeneous orientation of capture antibodies, making them a potential replacement for traditional microplates in various formats of ELISAs.
Development of an Anti-Methoxy Poly(ethylene glycol) (alpha-mPEG) Cell-Based Capture System to Measure mPEG and mPEGylated Molecules
(2014-10-14)
Chuang, Kuo-Hsiang
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Kao, Chien-Han
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Roffler, Steve R.
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Lu, Ssu-Jung
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Cheng, Ta-Chun
Quantitative pharmacokinetic analysis of methoxy-poly(ethylene glycol) (mPEG) and mPEGylated molecules is important for clinical drug development. Here we developed sensitive sandwich and competitive ELISAs by expressing an anti-mPEG antibody on the surface of fibroblasts for effective capture of mPEG molecules in biological samples. alpha-mPEG sandwich ELISA could quantify the higher-molecular-weight of mPEG (2, 5, and 20 kDa) and mPEGylated molecules. alpha-mPEG cell-based competitive ELISA was developed to measure the lower-molecular-weight of mPEG molecules (559, 750, and 1000 Da) at nanomolar levels. In addition, alpha-mPEG cell-based ELISA was unaffected by the presence of 10% human serum or murine serum. We further demonstrate that the alpha-mPEG cell-based ELISA determined similar pharmacokinetics of mPEG(5K) as traditional gamma counting of I-131-mPEG(5K). The alpha-mPEG cell-based ELISA may provide an accurate, high sensitivity and easy-to-use tool for directly measuring mPEG and mPEGylated molecules in complex biological samples to accelerate the clinical development of mPEG drugs.
Enhanced drug internalization and therapeutic efficacy of PEGylated nanoparticles by one-step formulation with anti-mPEG bispecific antibody in intrinsic drug-resistant breast cancer
(2019-08-01)
Cheng, Yi-An
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Chen, I-Ju
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Su, Yu-Cheng
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Cheng, Kai-Wen
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Lu, Yun-Chi
For those patients with HER2-overexpressing breast cancer, treatment with PEGylated liposomal doxorubicin (PLD) is inefficacious due to the intrinsic low sensitivity to doxorubicin. A very large increase in drug accumulation by active targeting may enhance the therapeutic efficacy of PLD. We established a humanized bispecific antibody (BsAb; mPEG x HER2) which has dual specificity for methoxy-polyethylene glycol (mPEG) and human epidermal growth factor receptor 2 (HER2) to enhance the specificity, internalization and anticancer activity of PLD for cancer cells that overexpress HER2. One-step formulation of PLD with mPEG x HER2 converted the PLD into HER2 targeted liposomes that were stable at 4 degrees C in PBS as well as at 37 degrees C in the presence of serum. alpha HER2/PLD induced receptor-mediated endocytosis and enhanced doxorubicin accumulation in MCF7/HER2 (HER2-amplified) breast cancer cells. alpha HER2/PLD also displayed more than 200-fold increased cytotoxicity to MCF7/HER2 cells and 28-fold increased cytotoxicity to drug-resistant MDA-MB-361 cells with a physical deletion of the TOP2A gene. alpha HER2/PLD specifically accumulated doxorubicin in the nucleus of cancer cells in tumor-bearing mice and produced significantly greater antitumor activity against MCF7/HER2 (P < 0.0001) and MDA-MB-361 (P < 0.05) tumors as compared to untargeted PLD. Furthermore, the cardiotoxicity of alpha HER2/PLD was similar to that of PLD in human cardiomyocytes and in mice. Our results indicate that the one-step formulation of PLD by mPEG x HER2 is a simple method to confer tumor specificity, increase drug internalization and enhance the anticancer activity of PLD against HER2-overexpressing and doxorubicin-resistant breast cancer.
Functional Production of a Soluble and Secreted Single-Chain Antibody by a Bacterial Secretion System
(2014-05-13)
Cheng, Chiu-Min
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Tzou, Shey-Cherng
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Zhuang, Ya-Han
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Huang, Chien-Chiao
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Kao, Chien-Han
Single-chain variable fragments (scFvs) serve as an alternative to full-length monoclonal antibodies used in research and therapeutic and diagnostic applications. However, when recombinant scFvs are overexpressed in bacteria, they often form inclusion bodies and exhibit loss of function. To overcome this problem, we developed an scFv secretion system in which scFv was fused with osmotically inducible protein Y (osmY), a bacterial secretory carrier protein, for efficient protein secretion. Anti-EGFR scFv (alpha EGFR) was fused with osmY (N- and C-termini) and periplasmic leader sequence (pelB) to generate alpha EGFR-osmY, osmY-alpha EGFR, and pelB-alpha EGFR (control), respectively. In comparison with the control, both the osmY-fused alpha EGFR scFvs were soluble and secreted into the LB medium. Furthermore, the yield of soluble alpha EGFR-osmY was 20-fold higher, and the amount of secreted protein was 250-fold higher than that of osmY-alpha EGFR. In addition, the antigenbinding activity of both the osmY-fused alpha EGFRs was 2-fold higher than that of the refolded pelB-alpha EGFR from inclusion bodies. Similar results were observed with alpha TAG72-osmY and alpha Her2-osmY. These results suggest that the N-terminus of osmY fused with scFv produces a high yield of soluble, functional, and secreted scFv, and the osmY-based bacterial secretion system may be used for the large-scale industrial production of low-cost alpha EGFRprotein.
Selective antibody activation through protease-activated pro-antibodies that mask binding sites with inhibitory domains
(2017-09-14)
Chen, I-Ju
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Chuang, Chih-Hung
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Hsieh, Yuan-Chin
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Lu, Yun-Chi
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Lin, Wen-Wei
Systemic injection of therapeutic antibodies may cause serious adverse effects due to on-target toxicity to the antigens expressed in normal tissues. To improve the targeting selectivity to the region of disease sites, we developed protease-activated pro-antibodies by masking the binding sites of antibodies with inhibitory domains that can be removed by proteases that are highly expressed at the disease sites. The latency-associated peptide (LAP), C2b or CBa of complement factor 2/B were linked, through a substrate peptide of matrix metalloproteinase-2 (MMP-2), to an anti-epidermal growth factor receptor (EGFR) antibody and an anti-tumor necrosis factor-alpha (TNF-alpha) antibody. Results showed that all the inhibitory domains could be removed by MMP-2 to restore the binding activities of the antibodies. LAP substantially reduced (53.8%) the binding activity of the anti-EGFR antibody on EGFR-expressing cells, whereas C2b and CBa were ineffective (21% and 9.3% reduction, respectively). Similarly, LAP also blocked 53.9% of the binding activity of the anti-TNF-alpha antibody. Finally, molecular dynamic simulation showed that the masking efficiency of LAP, C2b and CBa was 33.7%, 10.3% and -5.4%, respectively, over the binding sites of the antibodies. This strategy may aid in designing new protease-activated pro-antibodies that attain high therapeutic potency yet reduced systemic on-target toxicity.