Browsing by Author "Wang, Yeng-Tseng"

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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
;
Kao, Chien-Han
;
Roffler, Steve R.
;
Lu, Ssu-Jung
;
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.
Selective antibody activation through protease-activated pro-antibodies that mask binding sites with inhibitory domains
(2017-09-14)
Chen, I-Ju
;
Chuang, Chih-Hung
;
Hsieh, Yuan-Chin
;
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.