Browsing by Author "Su, Yu-Cheng"

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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.
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.
Premature Drug Release from Polyethylene Glycol (PEG)-Coated Liposomal Doxorubicin via Formation of the Membrane Attack Complex
(2020-07-28)
Chen, Even
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Chen, Bing-Mae
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Su, Yu-Cheng
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Chang, Yuan-Chih
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Cheng, Tian-Lu
Anti-polyethylene glycol (PEG) antibodies are present in many healthy individuals as well as in patients receiving polyethylene glycol-functionalized drugs. Antibodies against PEG-coated nanocarriers can accelerate their clearance, but their impact on nanodrug properties including nanocarrier integrity is unclear. Here, we show that anti-PEG IgG and IgM antibodies bind to PEG molecules on the surface of PEG-coated liposomal doxorubicin (Doxil, Doxisome, LC-101, and Lipo-Dox), resulting in complement activation, formation of the membrane attack complex (C51/-9) in the liposomal membrane, and rapid release of encapsulated doxorubicin from the liposomes. Drug release depended on both classical and alternative pathways of complement activation. Doxorubicin release of up to 40% was also observed in rats treated with anti-PEG IgG and PEG-coated liposomal doxorubicin. Our results demonstrate that anti-PEG antibodies can disrupt the membrane integrity of PEG-coated liposomal doxorubicin through activation of complement, which may alter therapeutic efficacy and safety in patients with high levels of pre-existing antibodies against PEG.
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.
Structural basis of polyethylene glycol recognition by antibody
(2020-01-07)
Lee, Cheng-Chung
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Su, Yu-Cheng
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Ko, Tzu-Ping
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Lin, Li-Ling
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Yang, Chih-Ya
Background Polyethylene glycol (PEG) is widely used in industry and medicine. Anti-PEG antibodies have been developed for characterizing PEGylated drugs and other applications. However, the underlying mechanism for specific PEG binding has not been elucidated. Methods The Fab of two cognate anti-PEG antibodies 3.3 and 2B5 were each crystallized in complex with PEG, and their structures were determined by X-ray diffraction. The PEG-Fab interactions in these two crystals were analyzed and compared with those in a PEG-containing crystal of an unrelated anti-hemagglutinin 32D6-Fab. The PEG-binding stoichiometry was examined by using analytical ultracentrifuge (AUC). Results A common PEG-binding mode to 3.3 and 2B5 is seen with an S-shaped core PEG fragment bound to two dyad-related Fab molecules. A nearby satellite binding site may accommodate parts of a longer PEG molecule. The core PEG fragment mainly interacts with the heavy-chain residues D31, W33, L102, Y103 and Y104, making extensive contacts with the aromatic side chains. At the center of each half-circle of the S-shaped PEG, a water molecule makes alternating hydrogen bonds to the ether oxygen atoms, in a similar configuration to that of a crown ether-bound lysine. Each satellite fragment is clamped between two arginine residues, R52 from the heavy chain and R29 from the light chain, and also interacts with several aromatic side chains. In contrast, the non-specifically bound PEG fragments in the 32D6-Fab crystal are located in the elbow region or at lattice contacts. The AUC data suggest that 3.3-Fab exists as a monomer in PEG-free solution but forms a dimer in the presence of PEG-550-MME, which is about the size of the S-shaped core PEG fragment. Conclusions The differing amino acids in 3.3 and 2B5 are not involved in PEG binding but engaged in dimer formation. In particular, the light-chain residue K53 of 2B5-Fab makes significant contacts with the other Fab in a dimer, whereas the corresponding N53 of 3.3-Fab does not. This difference in the protein-protein interaction between two Fab molecules in a dimer may explain the temperature dependence of 2B5 in PEG binding, as well as its inhibition by crown ether.
Synthesizable Injection-Locked Phase-Locked Loop with Multiphase Interlocking Digitally Controlled Oscillator Arrays
(2019-01-01)
Su, Yu-Cheng
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Chang, Kang-Yu
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Chin, Yu-Tung
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Chang, Chia-Wen
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Jou, Shyh-Jye
A synthesizable injection-locked phase-locked loop with multiphase interlocking digitally controlled oscillator arrays (SILPLL-IDCOs) is proposed. By doing interlocking of two digitally controlled oscillators (DCOs), the number of phase provided can be double and the oscillation frequency can be as fast as the original DCO. Moreover, it also adopts frequency tracking loop to isolate the injection path from the traditional PLL path in the ILPLL, so the race condition in the traditional ILPLL can be resolved. The chip has been designed and implemented in TSMC 40 nm GP 1P10M CMOS process technology. In the proposed synthesizable ILPLL, all logic cells and circuit components are using standard cell provided by foundry and our group. The total area of the synthesizable ILPLL core is only 0.01876 mm(2) and provides 8 phase output. The post-layout simulated RMS jitter from a 5.024 GHz output frequency is 0.048 %UI. The total measured power consumption is 10.97 mW at 5.024 GHz output frequency and 78.5 MHz reference clock.
Transient AID expression for in situ mutagenesis with improved cellular fitness
(2018-06-20)
Al-Qaisi, Talal Salem
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Su, Yu-Cheng
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Roffler, Steve R.
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生物科技學系
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Department of Biological Science and Technology
Activation induced cytidine deaminase (AID) in germinal center B cells introduces somatic DNA mutations in transcribed immunoglobulin genes to increase antibody diversity. Ectopic expression of AID coupled with selection has been successfully employed to develop proteins with desirable properties. However, this process is laborious and time consuming because many rounds of selection are typically required to isolate the target proteins. AID expression can also adversely affect cell viability due to off target mutagenesis. Here we compared stable and transient expression of AID mutants with different catalytic activities to determine conditions for maximum accumulation of mutations with minimal toxicity. We find that transient (3-5 days) expression of an AID upmutant in the presence of selection pressure could induce a high rate of mutagenesis in reporter genes without affecting cells growth and expansion. Our findings may help improve protein evolution by ectopic expression of AID and other enzymes that can induce DNA mutations.