Browsing by Author "Huang, Chien-Chiao"

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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
;
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.
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
;
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.