Browsing by Author "Kao, Chien-Han"

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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.
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.
Measurement of Poly(ethylene glycol) by Cell-Based Anti-poly(ethylene glycol) ELISA
(2010-03-15)
Chuang, Kuo-Hsiang
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Tzou, Shey-Cherng
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Cheng, Ta-Chun
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Kao, Chien-Han
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Tseng, Wei-Lung
Poly(ethylene glycol) (PEG) is increasingly used in clinical and experimental medicine. However, quantification of PEG and PEGylated small molecules remains laborious and unsatisfactory. In this report, we stably expressed a functional anti-PEG antibody on the surface of BALB 3T3 cells (3T3/alpha PEG cells) to develop a competitive enzyme-linked immunosorbent assay (ELISA) for PEG quantification. The alpha-PEG cell-coated plate bound biotinylated PEG(5K) (CH(3)-PEG(5K)-biotin) and CH(3)-PEG(5K)-(131)I more effectively than did a traditional anti-PEG antibody-coated plate. Competitive binding between PEG (2, 5, 10, or 20 kDa) and a known amount of CH(3)-PEG(5K)-biotin allowed construction of a reproducible competition curve. The alpha PEG cell-based competition ELISA measured small molecules derivatized by PEG(2K), PEG(5K), PEG(10K), PEG(20K), and PEG(5K) at concentrations as low as 58.6, 14.6, 3.7, 3.7, and 14.6 ng/mL respectively. Notably, the presence of serum or bovine serum albumin enhanced PEG measurement by the alpha PEG cell-based competition ELISA. Finally, we show here that the alpha PEG cell-based competition ELISA accurately delineated the pharmacokinetics of PEG(5K), comparable to those determined by direct measurement of radioactivity in blood after intravenous injection of CH(3)-PEG(5K)-(131)I into mice. This quantitative strategy may provide a simple and sensitive method for quantifying PEG and PEGylated small molecules in vivo.
Sensitivity of PEGylated Interferon Detection by Anti-Polyethylene Glycol (PEG) Antibodies Depends on PEG Length
(2013-08-01)
Cheng, Ta-Chun
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Chuang, Kuo-Hsiang
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Chen, Michael
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Wang, Hsin-Ell
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Tzou, Shey-Cherng
Attachment of poly(ethylene glycol) to proteins can mask immune epitopes to increase serum half-life, reduce immunogenicity, and enhance in vivo biological efficacy. However, PEGylation mediated epitope-masking may also limit sensitivity and accuracy of traditional ELISA. We previously described an anti-PEG-based sandwich ELISA for universal assay of PEGylated molecules. Here, we compared the quantitative assessment of PEGylated interferons by anti-PEG and traditional anti-interferon sandwich ELISA. The detection limits for PEG-Intron (12k-PEG) and Pegasys (40k-PEG) were 1.9 and 0.03 ng/mL for anti-PEG ELISA compared to 0.18 and 0.42 ng/mL for traditional anti-interferon sandwich ELISA. These results indicate that the anti-PEG sandwich ELISA was insensitive to PEGylation mediated epitope-masking and the sensitivity increased in proportion to the length of PEG. By contrast, PEG-masking interfered with detection by traditional anti-interferon sandwich ELISA. Human and mouse serum did not affect the sensitivity of anti-PEG ELISA but impeded traditional anti-interferon sandwich ELISA. The anti-PEG sandwich ELISA was comparable to anti-interferon sandwich ELISA and radioassay of I-131-Pegasys in pharmacokinetic studies in mice. The anti-PEG sandwich ELISA provides a sensitive, accurate, and convenient quantitative measurement of PEGylated protein drugs.