Ta-chun Cheng - Academia.edu (original) (raw)
Papers by Ta-chun Cheng
Pharmacological Research, 2022
The bidirectional interaction between carcinogens and gut microbiota that contributes to colorect... more The bidirectional interaction between carcinogens and gut microbiota that contributes to colorectal cancer is complicated. Reactivation of carcinogen metabolites by microbial β-glucuronidase (βG) in the gut potentially plays an important role in colorectal carcinogenesis. We assessed the chemoprotective effects and associated changes in gut microbiota induced by pre-administration of bacterial-specific βG inhibitor TCH-3511 in carcinogen azoxymethane (AOM)-treated APCMin/+ mice. AOM induced intestinal βG activity, which was reflected in increases in the incidence, formation, and number of tumors in the intestine. Notably, inhibition of gut microbial βG by TCH-3511 significantly reduced AOM-induced intestinal βG activity, decreased the number of polyps in both the small and large intestine to a frequency that was similar in mice without AOM exposure. AOM also led to lower diversity and altered composition in the gut microbiota with a significant increase in mucin-degrading Akkermansia genus. Conversely, mice treated with TCH-3511 and AOM exhibited a more similar gut microbiota structure as mice without AOM administration. Importantly, TCH-3511 treatment significant decreased Akkermansia genus and produced a concomitant increase in short-chain fatty acid butyrate-producing gut commensal microbes Lachnoospiraceae NK4A136 group genus in AOM-treated mice. Taken together, our results reveal a key role of gut microbial βG in promoting AOM-induced gut microbial dysbiosis and intestinal tumorigenesis, indicating the chemoprotective benefit of gut microbial βG inhibition against carcinogens via maintaining the gut microbiota balance and preventing cancer-associated gut microbial dysbiosis. Thus, the bacterial-specific βG inhibitor TCH-3511 is a potential chemoprevention agent for colorectal cancer.
The development of effective adjuvant is the key factor to boost the immunogenicity of tumor cell... more The development of effective adjuvant is the key factor to boost the immunogenicity of tumor cells as a tumor vaccine. In this study, we expressed membrane-bound granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-18 (IL-18) as adjuvants in tumor cells to stimulate immune response. B7 transmembrane domain fused GM-CSF and IL-18 was successfully expressed in the cell membrane and stimulated mouse splenocyte proliferation. Co-expression of GM-CSF and IL-18 reduced tumorigenesis (P<0.05) and enhanced tumor protective efficacy (P<0.05) significantly in comparison with GM-CSF alone. These results indicated that the combination of GM-CSF andIL-18 will enhance the immunogenicity of a cell-based anti-tumor vaccine. This membrane-bound approach can be applied to other cytokines for the development of novel vaccine strategies.
Abstract: Akt acts as a pivotal regulator in the PI3K/Akt signaling pathway and represents a pote... more Abstract: Akt acts as a pivotal regulator in the PI3K/Akt signaling pathway and represents a potential drug target for cancer therapy. To search for new inhibitors of Akt kinase, we performed a structure-based virtual screening using the DOCK 4.0 program and the X-ray crystal structure of human Akt kinase. From the virtual screening, 48 compounds were selected and subjected to the Akt kinase inhibition assay. Twenty-six of the test compounds showed more potent inhibitory effects on Akt kinase than the reference compound, H-89. These 26 compounds were further evaluated for their cytotoxicity against HCT-116 human colon cancer cells and HEK-293 normal human embryonic kidney cells. Twelve compounds were found to display more potent or comparable cytotoxic activity compared
Pharmacological Research, 2018
Irinotecan (CPT-11), a first-line chemotherapy for advanced colorectal cancer, causes serious dia... more Irinotecan (CPT-11), a first-line chemotherapy for advanced colorectal cancer, causes serious diarrhea in patients receiving treatment. The underlying mechanism has been shown that the active metabolite of CPT-11, SN-38, is metabolized to the inactive metabolite SN-38 glucuronide (SN-38G) during hepatic glucuronidation, and subsequently is exported into the intestine, where SN-38G is hydrolyzed by bacterial -glucuronidase (G) to be SN-38, thus leading to intestinal toxicity. Thus, inhibition of the intestinal bacterial G activity is expected to prevent CPT-11-induced diarrhea. However, the effects of such inhibition on serum pharmacokinetics of SN-38, the key determinant of CPT-11 treatment, are uncertain. Here, we determined the effects of a potent E. coli G (eG)-specific inhibitor pyrazolo[4,3-c]quinoline derivative (TCH-3562) for potential use in tumor therapy. TCH-3562 exhibited efficacious inhibitory potency of endogenous G activity in two anaerobes, Eubacterium sp. and Peptostreptococcus anaerobius. Oral administration of TCH-3562 also effectively reduced the bacterial G activity in mice intestine. Moreover, pharmacokinetic analysis of TCH-3562 revealed a relatively low amount of TCH-3562 was detected in the plasma whereas the majority of TCH-3562 was found in the feces. Importantly, co-treatment of CPT-11 and TCH-3562 did not decrease active SN-38 level in mice plasma. Finally, we established that TCH-3562 as an adjuvant treatment showed protective effects on CPT-11-induced diarrhea and had no negative effects on the therapeutic efficacy of CPT-11 in tumor-bearing mice. Therefore, inhibiting of the intestinal bacterial G activity by the specific inhibitor, TCH-3562, is promising to prevent CPT-11-induced diarrhea while maintaining CPT-11 anti-tumor efficacy that may have clinical potentials for the treatment with CPT-11.
Journal of Medicinal Chemistry, 2017
The direct inhibition of bacterial β-glucuronidase (βG) activity is expected to reduce the reacti... more The direct inhibition of bacterial β-glucuronidase (βG) activity is expected to reduce the reactivation of glucuronideconjugated drugs in the intestine, thereby reducing drug toxicity. In this study, we report on the effects of pyrazolo[4,3-c] quinolines acting as a new class of bacterial βG-specific inhibitors in a pH-dependent manner. Refinement of this chemotype for establishing structure−activity relationship resulted in the identification of potential leads. Notably, the oral administration of 3-amino-4-(4-fluorophenylamino)-1H-pyrazolo[4,3-c]quinoline (42) combined with chemotherapeutic CPT-11 treatment prevented CPT-11-induced serious diarrhea while maintaining the antitumor efficacy in tumor-bearing mice. Importantly, the inhibitory effects of 42 to E. coli βG was reduced as the pH decreased due to the various surface charges of the active pocket of the enzyme, which may make their combination more favorable at neutral pH. These results demonstrate novel insights into the potent bacterial βG-specific inhibitor that would allow this inhibitor to be used for the purpose of reducing drug toxicity.
Scientific Reports, 2017
Systemic injection of therapeutic antibodies may cause serious adverse effects due to on-target t... more 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-α (TNF-α) 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-α 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 proantibodies that attain high therapeutic potency yet reduced systemic on-target toxicity. Many monoclonal antibodies have been approved for treating malignant cancers 1-3 , inflammatory diseases such as rheumatoid arthritis 4, 5 and osteoporosis 6. According to a recent survey, about 50 monoclonal antibodies are currently approved for clinical use and approximately 350 monoclonal antibodies are under development 7. Unlike many small molecule drugs that may target multiple proteins, monoclonal antibodies typically target specific molecules (antigens) associated with diseases. Conceptually, monoclonal antibodies should display higher specificity and have fewer systemic side effects than small molecule drugs. In some cases, however, the antigens targeted by therapeutic antibodies are not expressed exclusively at the disease sites. Systemic injection of therapeutic monoclonal antibodies may cause considerable adverse effects 8 and thus decrease treatment efficacies. For example, epidermal growth factor receptor (EGFR) is over-expressed in some tumor cells and plays an important role in tumor progression 9. However, EGFR is also expressed in some epithelial cells 10, 11. Systemic injection of anti-EGFR antibodies (Erbitux) is known to induce adverse effects such
Scientific reports, Jan 9, 2017
Intestinal bacterial β-glucuronidase (βG) hydrolyzes glucuronidated metabolites to their toxic fo... more Intestinal bacterial β-glucuronidase (βG) hydrolyzes glucuronidated metabolites to their toxic form in intestines, resulting in intestinal damage. The development of a method to inhibit βG is thus important but has been limited by the difficulty of directly assessing enzyme activity in live animals. Here, we utilized a fluorescent probe, fluorescein di-β-D-glucuronide (FDGlcU), to non-invasively image the intestinal bacterial βG activity in nude mice. In vitro cell-based assays showed that the detection limit is 10(4) colony-forming units/well of βG-expressing bacteria, and that 7.81 ng/mL of FDGlcU is enough to generate significant fluorescent signal. In whole-body optical images of nude mice, the maximum fluorescence signal for βG activity in intestines was detected 3 hours after gavage with FDGlcU. Following pretreatment with a bacterial βG inhibitor, the fluorescence signal was significantly reduced in abdomens and excised intestines images. For a 4-day antibiotic treatment to d...
Analytical chemistry, Jan 20, 2016
Sensitive determination of the pharmacokinetics of PEGylated molecules can accelerate the process... more Sensitive determination of the pharmacokinetics of PEGylated molecules can accelerate the process of drug development. Here, we combined different anti-PEG Fab expressing 293T cells as capture cells (293T/3.3, 293T/6.3, and 293T/15-2b cells) with four detective anti-PEG antibodies (3.3, 6.3, 7A4, or 15-2b) to optimize an anti-PEG cell-based sandwich ELISA. Then, we quantified free PEG (mPEG2K-NH2 and mPEG5K-NH2) or PEG-conjugated small molecules (mPEG5K-biotin and mPEG5K-NIR797), proteins (PegIntron and Pegasys), and nanoparticles (Liposomal-Doxorubicin and quantum-dots). The combination of 293T/15-2b cells and the 7A4 detection antibody was best sensitivity for free PEG, PEG-like molecules, and PEGylated proteins with detection at ng mL(-1) levels. On the other hand, 293T/3.3 cells combined with the 15-2b antibody had the highest sensitivity for quantifying Lipo-Dox at 2 ng mL(-1). All three types of anti-PEG cells combined with the 15-2b antibody had high sensitivity for quantum d...
Journal of Biomedical Nanotechnology, 2017
Solid lipid nanoparticles (SLNs) are suitable candidates for the delivery of various anti-cancer ... more Solid lipid nanoparticles (SLNs) are suitable candidates for the delivery of various anti-cancer drugs. However, currently insufficient tumor-permeability and non-specific uptake by the reticuloendothelial system limits the application of SLNs. Here, we developed novel pH-sensitive cationic polyoxyethylene (PEGylated) SLNs (PEG-SLNs +) that could accumulate long-term at various tumor sites to enhance the therapeutic efficiency of camptothecin (CPT). These CPT-loaded PEG-SLNs + (CPT-PEG-SLNs +) were spherical nanoparticles, with small size (∼52.3 ± 1.7 nm), positive charge (∼34.3 ± 3.5 mV) and high entrapment efficiency (∼99.4 ± 1.7%). Drug release profile indicated the overall released amount of CPT from CPT-PEG-SLNs + at pH 5.5 was 20.2% more than at pH 7.4, suggesting CPT-PEG-SLNs + were a pH-sensitive SLNs. This PEG-SLNs + could be efficiently uptaken into cells to inhibit the proliferation of CL1-5 cells (IC 50 = 0 37 ± 0 21 ug/ml) or HCC36 cells (IC 50 = 0 16 ± 0 43 ug/ml). In living animal, our PEG-SLNs + could accumulate long-term (for more than 120 hours) in various types of tumor, including human lung carcinoma (NCI-H358, CRL5802, CL1-5), human colon carcinoma (HCT-116) and human hepatocellular carcinoma (HCC36), and CPT-PEG-SLNs + could efficiently enhance the therapeutic efficiency of CPT to suppress the growth of the HCC36 or CL1-5 tumors. Therefore, Successful development of these pH-sensitive PEGylated cationic SLNs may provide a selective and efficient drug delivery system for cancer therapy.
PloS one, 2016
Molecular weight markers that can tolerate denaturing conditions and be auto-detected by secondar... more Molecular weight markers that can tolerate denaturing conditions and be auto-detected by secondary antibodies offer great efficacy and convenience for Western Blotting. Here, we describe M&R LE protein markers which contain linear epitopes derived from the heavy chain constant regions of mouse and rabbit immunoglobulin G (IgG Fc LE). These markers can be directly recognized and stained by a wide range of anti-mouse and anti-rabbit secondary antibodies. We selected three mouse (M1, M2 and M3) linear IgG1 and three rabbit (R1, R2 and R3) linear IgG heavy chain epitope candidates based on their respective crystal structures. Western blot analysis indicated that M2 and R2 linear epitopes are effectively recognized by anti-mouse and anti-rabbit secondary antibodies, respectively. We fused the M2 and R2 epitopes (M&R LE) and incorporated the polypeptide in a range of 15-120 kDa auto-detecting markers (M&R LE protein marker). The M&R LE protein marker can be auto-detected by anti-mouse and...
Scientific Reports, 2016
Attachment of polyethylene glycol (PEG) molecules to nanoparticles (PEGylation) is a widely-used ... more Attachment of polyethylene glycol (PEG) molecules to nanoparticles (PEGylation) is a widely-used method to improve the stability, biocompatibility and half-life of nanomedicines. However, the evaluation of the PEGylated nanomedicine pharmacokinetics (PK) requires the decomposition of particles and purification of lead compounds before analysis by high performance liquid chromatography (HPLC), mass spectrometry, etc. Therefore, a method to directly quantify undecomposed PEGylated nanoparticles is needed. In this study, we developed anti-PEG bioparticles and combined them with anti-PEG antibodies to generate a quantitative enzyme-linked immunosorbent assay (ELISA) for direct measurement of PEGylated nanoparticles without compound purification. The anti-PEG bioparticles quantitative ELISA directly quantify PEG-quantum dots (PEG-QD), PEGstabilizing super-paramagnetic iron oxide (PEG-SPIO), Lipo-Dox and PEGASYS and the detection limits were 0.01 nM, 0.1 nM, 15.63 ng/mL and 0.48 ng/mL, respectively. Furthermore, this anti-PEG bioparticle-based ELISA tolerated samples containing up to 10% mouse or human serum. There was no significant difference in pharmacokinetic studies of radiolabeled PEG-nanoparticles (Nano-X-111 In) through anti-PEG bioparticle-based ELISA and a traditional gamma counter. These results suggest that the anti-PEG bioparticle-based ELISA may provide a direct and effective method for the quantitation of any whole PEGylated nanoparticles without sample preparation. PEGylation of nanoparticles may improve their biocompatibility, reduce immunogenicity and enhance their half-life in the human body. PEGylated nanoparticles are widely used and have been developed into various types of nanomedicine. For example, PEG-modified liposomal doxorubicin (Caelyx and Lipo-Dox) has been used to treat ovarian, breast carcinomas and Kaposi's sarcoma 1,2. PEGylated Interferon (Pegasys 3,4 , PEG-Intron 5) was employed as a long-term therapeutic agent for hepatitis C. Several PEGylated polymeric micelle formulations, such as Paclitaxel and Cisplatin, are currently in phase I/II clinical trials for treatment of stomach cancer and solid tumors 6,7. PEG-modified imaging nanoparticles, such as quantum dots (QD) 8 and clinically approved super-paramagnetic iron oxide (PEG-SPIO) 9 have also been used to track the localization of tumors by optical
PLOS ONE, 2015
The development of effective adjuvant is the key factor to boost the immunogenicity of tumor cell... more The development of effective adjuvant is the key factor to boost the immunogenicity of tumor cells as a tumor vaccine. In this study, we expressed membrane-bound granulocytemacrophage colony-stimulating factor (GM-CSF) and interleukin-18 (IL-18) as adjuvants in tumor cells to stimulate immune response. B7 transmembrane domain fused GM-CSF and IL-18 was successfully expressed in the cell membrane and stimulated mouse splenocyte proliferation. Co-expression of GM-CSF and IL-18 reduced tumorigenesis (P<0.05) and enhanced tumor protective efficacy (P<0.05) significantly in comparison with GM-CSF alone. These results indicated that the combination of GM-CSF andIL-18 will enhance the immunogenicity of a cell-based anti-tumor vaccine. This membrane-bound approach can be applied to other cytokines for the development of novel vaccine strategies.
Veterinary journal (London, England : 1997), Jan 14, 2015
The envelope glycoprotein E2 of classical swine fever virus (CSFV) is widely used as a marker for... more The envelope glycoprotein E2 of classical swine fever virus (CSFV) is widely used as a marker for measuring vaccine efficacy and antibody titer. The glycosylation profile of E2 may affect the immunogenicity of the vaccine and the timing of re-vaccination. In this study, a human embryonic kidney cell line was used to secrete fully-glycosylated CSFV E2, which was then coated onto ELISA plates without purification or adjustment. The resulting E2-secreting medium-direct-coating (E2-mDc) ELISA was successfully used to measure anti-E2 antibody titers in vaccinated and field pig sera samples. Compared with a virus neutralization test (as standard), the E2-mDc ELISA was found to be more accurate (90%) than a commercial CSFV antibody diagnostic kit (62%). In conclusion, the mammalian cell-secreted antigen can provide cheap, accurate and effective assays for vaccine efficacy and disease diagnoses.
TheScientificWorldJournal, 2015
Glucuronidation is a major metabolism process of detoxification for carcinogens, 4-(methylnitrosa... more Glucuronidation is a major metabolism process of detoxification for carcinogens, 4-(methylnitrosamino)-1-(3-pyridy)-1-butanone (NNK) and 1,2-dimethylhydrazine (DMH), of reactive oxygen species (ROS). However, intestinal E. coli β-glucuronidase (eβG) has been considered pivotal to colorectal carcinogenesis. Specific inhibition of eβG may prevent reactivating the glucuronide-carcinogen and protect the intestine from ROS-mediated carcinogenesis. In order to develop specific eβG inhibitors, we found that 59 candidate compounds obtained from the initial virtual screening had high inhibition specificity against eβG but not human βG. In particular, we found that compounds 7145 and 4041 with naphthalenylidene-benzenesulfonamide (NYBS) are highly effective and selective to inhibit eβG activity. Compound 4041 (IC50 = 2.8 μM) shows a higher inhibiting ability than compound 7145 (IC50 = 31.6 μM) against eβG. Furthermore, the molecular docking analysis indicates that compound 4041 has two hy...
International journal of molecular sciences, 2015
Akt acts as a pivotal regulator in the PI3K/Akt signaling pathway and represents a potential drug... more Akt acts as a pivotal regulator in the PI3K/Akt signaling pathway and represents a potential drug target for cancer therapy. To search for new inhibitors of Akt kinase, we performed a structure-based virtual screening using the DOCK 4.0 program and the X-ray crystal structure of human Akt kinase. From the virtual screening, 48 compounds were selected and subjected to the Akt kinase inhibition assay. Twenty-six of the test compounds showed more potent inhibitory effects on Akt kinase than the reference compound, H-89. These 26 compounds were further evaluated for their cytotoxicity against HCT-116 human colon cancer cells and HEK-293 normal human embryonic kidney cells. Twelve compounds were found to display more potent or comparable cytotoxic activity compared to compound H-89 against HCT-116 colon cancer cells. The best results were obtained with Compounds a46 and a48 having IC50 values (for HCT-116) of 11.1 and 9.5 µM, respectively, and selectivity indices (IC50 for HEK-293/IC50 f...
Molecular cancer therapeutics, 2014
Beta-glucuronidase (βG) is a potential biomarker for cancer diagnosis and prodrug therapy. The ab... more Beta-glucuronidase (βG) is a potential biomarker for cancer diagnosis and prodrug therapy. The ability to image βG activity in patients would assist in personalized glucuronide prodrug cancer therapy. However, whole-body imaging of βG activity for medical usage is not yet available. Here, we developed a radioactive βG activity-based trapping probe for positron emission tomography (PET). We generated a (124)I-tyramine-conjugated difluoromethylphenol beta-glucuronide probe (TrapG) to form (124)I-TrapG that could be selectively activated by βG for subsequent attachment of (124)I-tyramine to nucleophilic moieties near βG-expressing sites. We estimated the specificity of a fluorescent FITC-TrapG, the cytotoxicity of tyramine-TrapG, and the serum half-life of (124)I-TrapG. βG targeting of (124)I-TrapG in vivo was examined by micro-PET. The biodistribution of (131)I-TrapG was investigated in different organs. Finally, we imaged the endogenous βG activity and assessed its correlation with t...
Biomaterials, 2014
Methoxy PEGylated nanoparticles (mPEG-NPs) are increasingly used for cancer imaging and therapy. ... more Methoxy PEGylated nanoparticles (mPEG-NPs) are increasingly used for cancer imaging and therapy. Here we describe a general and simple approach to confer tumor tropism to any mPEG-NP. We demonstrate this approach with humanized bispecific antibodies (BsAbs) that can bind to both mPEG molecules on mPEG-NPs and to EGFR or HER2 molecules overexpressed on the surface of cancer cells. Simple mixing of BsAbs with mPEG-NPs can mediate preferential binding of diverse mPEG-NPs to cancer cells that overexpress EGFR or HER2 under physiological conditions and significantly increase cancer cell killing by liposomal doxorubicin to EGFR þ and HER2 þ cancer cells. BsAbs modification also enhanced accumulation of fluorescence-labeled NPs and significantly increased the anticancer activity of drugloaded NPs to antigen-positive human tumors in a mouse model. Anti-mPEG BsAbs offer a simple one-step method to confer tumor specificity to mPEG-NPs for enhanced tumor accumulation and improved therapeutic efficacy.
Macromolecules, 2014
Quantitative pharmacokinetic analysis of methoxy-poly(ethylene glycol) (mPEG) and mPEGylated mole... more 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. α-mPEG sandwich ELISA could quantify the highermolecular-weight of mPEG (2, 5, and 20 kDa) and mPEGylated molecules. α-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, α-mPEG cell-based ELISA was unaffected by the presence of 10% human serum or murine serum. We further demonstrate that the α-mPEG cell-based ELISA determined similar pharmacokinetics of mPEG 5K as traditional gamma counting of 131 I-mPEG 5K. The α-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. ■ INTRODUCTION PEGylation is a FDA-approved technology for enhancing the bioavailability, safety, stability, and efficacy of a wide range of small molecules, proteins, liposomes, or nanoparticles. 1,2 The monomethoxylated form of PEG (mPEG) is widely used for clinical drug modification 3,4 because mPEG modification can reduce nonspecific binding and prevent recognition by the reticuloendothelial system (RES) in vivo. 5,6 Hence, a wide range
Bioconjugate Chemistry, 2013
Attachment of poly(ethylene glycol) to proteins can mask immune epitopes to increase serum half-l... more 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 antiinterferon 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 131 I-Pegasys in pharmacokinetic studies in mice. The anti-PEG sandwich ELISA provides a sensitive, accurate, and convenient quantitative measurement of PEGylated protein drugs.
PLoS ONE, 2014
Single-chain variable fragments (scFvs) serve as an alternative to full-length monoclonal antibod... more 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 (aEGFR) was fused with osmY (N-and C-termini) and periplasmic leader sequence (pelB) to generate aEGFR-osmY, osmY-aEGFR, and pelB-aEGFR (control), respectively. In comparison with the control, both the osmYfused aEGFR scFvs were soluble and secreted into the LB medium. Furthermore, the yield of soluble aEGFR-osmY was 20fold higher, and the amount of secreted protein was 250-fold higher than that of osmY-aEGFR. In addition, the antigenbinding activity of both the osmY-fused aEGFRs was 2-fold higher than that of the refolded pelB-aEGFR from inclusion bodies. Similar results were observed with aTAG72-osmY and aHer2-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 aEGFR protein.
Pharmacological Research, 2022
The bidirectional interaction between carcinogens and gut microbiota that contributes to colorect... more The bidirectional interaction between carcinogens and gut microbiota that contributes to colorectal cancer is complicated. Reactivation of carcinogen metabolites by microbial β-glucuronidase (βG) in the gut potentially plays an important role in colorectal carcinogenesis. We assessed the chemoprotective effects and associated changes in gut microbiota induced by pre-administration of bacterial-specific βG inhibitor TCH-3511 in carcinogen azoxymethane (AOM)-treated APCMin/+ mice. AOM induced intestinal βG activity, which was reflected in increases in the incidence, formation, and number of tumors in the intestine. Notably, inhibition of gut microbial βG by TCH-3511 significantly reduced AOM-induced intestinal βG activity, decreased the number of polyps in both the small and large intestine to a frequency that was similar in mice without AOM exposure. AOM also led to lower diversity and altered composition in the gut microbiota with a significant increase in mucin-degrading Akkermansia genus. Conversely, mice treated with TCH-3511 and AOM exhibited a more similar gut microbiota structure as mice without AOM administration. Importantly, TCH-3511 treatment significant decreased Akkermansia genus and produced a concomitant increase in short-chain fatty acid butyrate-producing gut commensal microbes Lachnoospiraceae NK4A136 group genus in AOM-treated mice. Taken together, our results reveal a key role of gut microbial βG in promoting AOM-induced gut microbial dysbiosis and intestinal tumorigenesis, indicating the chemoprotective benefit of gut microbial βG inhibition against carcinogens via maintaining the gut microbiota balance and preventing cancer-associated gut microbial dysbiosis. Thus, the bacterial-specific βG inhibitor TCH-3511 is a potential chemoprevention agent for colorectal cancer.
The development of effective adjuvant is the key factor to boost the immunogenicity of tumor cell... more The development of effective adjuvant is the key factor to boost the immunogenicity of tumor cells as a tumor vaccine. In this study, we expressed membrane-bound granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-18 (IL-18) as adjuvants in tumor cells to stimulate immune response. B7 transmembrane domain fused GM-CSF and IL-18 was successfully expressed in the cell membrane and stimulated mouse splenocyte proliferation. Co-expression of GM-CSF and IL-18 reduced tumorigenesis (P<0.05) and enhanced tumor protective efficacy (P<0.05) significantly in comparison with GM-CSF alone. These results indicated that the combination of GM-CSF andIL-18 will enhance the immunogenicity of a cell-based anti-tumor vaccine. This membrane-bound approach can be applied to other cytokines for the development of novel vaccine strategies.
Abstract: Akt acts as a pivotal regulator in the PI3K/Akt signaling pathway and represents a pote... more Abstract: Akt acts as a pivotal regulator in the PI3K/Akt signaling pathway and represents a potential drug target for cancer therapy. To search for new inhibitors of Akt kinase, we performed a structure-based virtual screening using the DOCK 4.0 program and the X-ray crystal structure of human Akt kinase. From the virtual screening, 48 compounds were selected and subjected to the Akt kinase inhibition assay. Twenty-six of the test compounds showed more potent inhibitory effects on Akt kinase than the reference compound, H-89. These 26 compounds were further evaluated for their cytotoxicity against HCT-116 human colon cancer cells and HEK-293 normal human embryonic kidney cells. Twelve compounds were found to display more potent or comparable cytotoxic activity compared
Pharmacological Research, 2018
Irinotecan (CPT-11), a first-line chemotherapy for advanced colorectal cancer, causes serious dia... more Irinotecan (CPT-11), a first-line chemotherapy for advanced colorectal cancer, causes serious diarrhea in patients receiving treatment. The underlying mechanism has been shown that the active metabolite of CPT-11, SN-38, is metabolized to the inactive metabolite SN-38 glucuronide (SN-38G) during hepatic glucuronidation, and subsequently is exported into the intestine, where SN-38G is hydrolyzed by bacterial -glucuronidase (G) to be SN-38, thus leading to intestinal toxicity. Thus, inhibition of the intestinal bacterial G activity is expected to prevent CPT-11-induced diarrhea. However, the effects of such inhibition on serum pharmacokinetics of SN-38, the key determinant of CPT-11 treatment, are uncertain. Here, we determined the effects of a potent E. coli G (eG)-specific inhibitor pyrazolo[4,3-c]quinoline derivative (TCH-3562) for potential use in tumor therapy. TCH-3562 exhibited efficacious inhibitory potency of endogenous G activity in two anaerobes, Eubacterium sp. and Peptostreptococcus anaerobius. Oral administration of TCH-3562 also effectively reduced the bacterial G activity in mice intestine. Moreover, pharmacokinetic analysis of TCH-3562 revealed a relatively low amount of TCH-3562 was detected in the plasma whereas the majority of TCH-3562 was found in the feces. Importantly, co-treatment of CPT-11 and TCH-3562 did not decrease active SN-38 level in mice plasma. Finally, we established that TCH-3562 as an adjuvant treatment showed protective effects on CPT-11-induced diarrhea and had no negative effects on the therapeutic efficacy of CPT-11 in tumor-bearing mice. Therefore, inhibiting of the intestinal bacterial G activity by the specific inhibitor, TCH-3562, is promising to prevent CPT-11-induced diarrhea while maintaining CPT-11 anti-tumor efficacy that may have clinical potentials for the treatment with CPT-11.
Journal of Medicinal Chemistry, 2017
The direct inhibition of bacterial β-glucuronidase (βG) activity is expected to reduce the reacti... more The direct inhibition of bacterial β-glucuronidase (βG) activity is expected to reduce the reactivation of glucuronideconjugated drugs in the intestine, thereby reducing drug toxicity. In this study, we report on the effects of pyrazolo[4,3-c] quinolines acting as a new class of bacterial βG-specific inhibitors in a pH-dependent manner. Refinement of this chemotype for establishing structure−activity relationship resulted in the identification of potential leads. Notably, the oral administration of 3-amino-4-(4-fluorophenylamino)-1H-pyrazolo[4,3-c]quinoline (42) combined with chemotherapeutic CPT-11 treatment prevented CPT-11-induced serious diarrhea while maintaining the antitumor efficacy in tumor-bearing mice. Importantly, the inhibitory effects of 42 to E. coli βG was reduced as the pH decreased due to the various surface charges of the active pocket of the enzyme, which may make their combination more favorable at neutral pH. These results demonstrate novel insights into the potent bacterial βG-specific inhibitor that would allow this inhibitor to be used for the purpose of reducing drug toxicity.
Scientific Reports, 2017
Systemic injection of therapeutic antibodies may cause serious adverse effects due to on-target t... more 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-α (TNF-α) 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-α 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 proantibodies that attain high therapeutic potency yet reduced systemic on-target toxicity. Many monoclonal antibodies have been approved for treating malignant cancers 1-3 , inflammatory diseases such as rheumatoid arthritis 4, 5 and osteoporosis 6. According to a recent survey, about 50 monoclonal antibodies are currently approved for clinical use and approximately 350 monoclonal antibodies are under development 7. Unlike many small molecule drugs that may target multiple proteins, monoclonal antibodies typically target specific molecules (antigens) associated with diseases. Conceptually, monoclonal antibodies should display higher specificity and have fewer systemic side effects than small molecule drugs. In some cases, however, the antigens targeted by therapeutic antibodies are not expressed exclusively at the disease sites. Systemic injection of therapeutic monoclonal antibodies may cause considerable adverse effects 8 and thus decrease treatment efficacies. For example, epidermal growth factor receptor (EGFR) is over-expressed in some tumor cells and plays an important role in tumor progression 9. However, EGFR is also expressed in some epithelial cells 10, 11. Systemic injection of anti-EGFR antibodies (Erbitux) is known to induce adverse effects such
Scientific reports, Jan 9, 2017
Intestinal bacterial β-glucuronidase (βG) hydrolyzes glucuronidated metabolites to their toxic fo... more Intestinal bacterial β-glucuronidase (βG) hydrolyzes glucuronidated metabolites to their toxic form in intestines, resulting in intestinal damage. The development of a method to inhibit βG is thus important but has been limited by the difficulty of directly assessing enzyme activity in live animals. Here, we utilized a fluorescent probe, fluorescein di-β-D-glucuronide (FDGlcU), to non-invasively image the intestinal bacterial βG activity in nude mice. In vitro cell-based assays showed that the detection limit is 10(4) colony-forming units/well of βG-expressing bacteria, and that 7.81 ng/mL of FDGlcU is enough to generate significant fluorescent signal. In whole-body optical images of nude mice, the maximum fluorescence signal for βG activity in intestines was detected 3 hours after gavage with FDGlcU. Following pretreatment with a bacterial βG inhibitor, the fluorescence signal was significantly reduced in abdomens and excised intestines images. For a 4-day antibiotic treatment to d...
Analytical chemistry, Jan 20, 2016
Sensitive determination of the pharmacokinetics of PEGylated molecules can accelerate the process... more Sensitive determination of the pharmacokinetics of PEGylated molecules can accelerate the process of drug development. Here, we combined different anti-PEG Fab expressing 293T cells as capture cells (293T/3.3, 293T/6.3, and 293T/15-2b cells) with four detective anti-PEG antibodies (3.3, 6.3, 7A4, or 15-2b) to optimize an anti-PEG cell-based sandwich ELISA. Then, we quantified free PEG (mPEG2K-NH2 and mPEG5K-NH2) or PEG-conjugated small molecules (mPEG5K-biotin and mPEG5K-NIR797), proteins (PegIntron and Pegasys), and nanoparticles (Liposomal-Doxorubicin and quantum-dots). The combination of 293T/15-2b cells and the 7A4 detection antibody was best sensitivity for free PEG, PEG-like molecules, and PEGylated proteins with detection at ng mL(-1) levels. On the other hand, 293T/3.3 cells combined with the 15-2b antibody had the highest sensitivity for quantifying Lipo-Dox at 2 ng mL(-1). All three types of anti-PEG cells combined with the 15-2b antibody had high sensitivity for quantum d...
Journal of Biomedical Nanotechnology, 2017
Solid lipid nanoparticles (SLNs) are suitable candidates for the delivery of various anti-cancer ... more Solid lipid nanoparticles (SLNs) are suitable candidates for the delivery of various anti-cancer drugs. However, currently insufficient tumor-permeability and non-specific uptake by the reticuloendothelial system limits the application of SLNs. Here, we developed novel pH-sensitive cationic polyoxyethylene (PEGylated) SLNs (PEG-SLNs +) that could accumulate long-term at various tumor sites to enhance the therapeutic efficiency of camptothecin (CPT). These CPT-loaded PEG-SLNs + (CPT-PEG-SLNs +) were spherical nanoparticles, with small size (∼52.3 ± 1.7 nm), positive charge (∼34.3 ± 3.5 mV) and high entrapment efficiency (∼99.4 ± 1.7%). Drug release profile indicated the overall released amount of CPT from CPT-PEG-SLNs + at pH 5.5 was 20.2% more than at pH 7.4, suggesting CPT-PEG-SLNs + were a pH-sensitive SLNs. This PEG-SLNs + could be efficiently uptaken into cells to inhibit the proliferation of CL1-5 cells (IC 50 = 0 37 ± 0 21 ug/ml) or HCC36 cells (IC 50 = 0 16 ± 0 43 ug/ml). In living animal, our PEG-SLNs + could accumulate long-term (for more than 120 hours) in various types of tumor, including human lung carcinoma (NCI-H358, CRL5802, CL1-5), human colon carcinoma (HCT-116) and human hepatocellular carcinoma (HCC36), and CPT-PEG-SLNs + could efficiently enhance the therapeutic efficiency of CPT to suppress the growth of the HCC36 or CL1-5 tumors. Therefore, Successful development of these pH-sensitive PEGylated cationic SLNs may provide a selective and efficient drug delivery system for cancer therapy.
PloS one, 2016
Molecular weight markers that can tolerate denaturing conditions and be auto-detected by secondar... more Molecular weight markers that can tolerate denaturing conditions and be auto-detected by secondary antibodies offer great efficacy and convenience for Western Blotting. Here, we describe M&R LE protein markers which contain linear epitopes derived from the heavy chain constant regions of mouse and rabbit immunoglobulin G (IgG Fc LE). These markers can be directly recognized and stained by a wide range of anti-mouse and anti-rabbit secondary antibodies. We selected three mouse (M1, M2 and M3) linear IgG1 and three rabbit (R1, R2 and R3) linear IgG heavy chain epitope candidates based on their respective crystal structures. Western blot analysis indicated that M2 and R2 linear epitopes are effectively recognized by anti-mouse and anti-rabbit secondary antibodies, respectively. We fused the M2 and R2 epitopes (M&R LE) and incorporated the polypeptide in a range of 15-120 kDa auto-detecting markers (M&R LE protein marker). The M&R LE protein marker can be auto-detected by anti-mouse and...
Scientific Reports, 2016
Attachment of polyethylene glycol (PEG) molecules to nanoparticles (PEGylation) is a widely-used ... more Attachment of polyethylene glycol (PEG) molecules to nanoparticles (PEGylation) is a widely-used method to improve the stability, biocompatibility and half-life of nanomedicines. However, the evaluation of the PEGylated nanomedicine pharmacokinetics (PK) requires the decomposition of particles and purification of lead compounds before analysis by high performance liquid chromatography (HPLC), mass spectrometry, etc. Therefore, a method to directly quantify undecomposed PEGylated nanoparticles is needed. In this study, we developed anti-PEG bioparticles and combined them with anti-PEG antibodies to generate a quantitative enzyme-linked immunosorbent assay (ELISA) for direct measurement of PEGylated nanoparticles without compound purification. The anti-PEG bioparticles quantitative ELISA directly quantify PEG-quantum dots (PEG-QD), PEGstabilizing super-paramagnetic iron oxide (PEG-SPIO), Lipo-Dox and PEGASYS and the detection limits were 0.01 nM, 0.1 nM, 15.63 ng/mL and 0.48 ng/mL, respectively. Furthermore, this anti-PEG bioparticle-based ELISA tolerated samples containing up to 10% mouse or human serum. There was no significant difference in pharmacokinetic studies of radiolabeled PEG-nanoparticles (Nano-X-111 In) through anti-PEG bioparticle-based ELISA and a traditional gamma counter. These results suggest that the anti-PEG bioparticle-based ELISA may provide a direct and effective method for the quantitation of any whole PEGylated nanoparticles without sample preparation. PEGylation of nanoparticles may improve their biocompatibility, reduce immunogenicity and enhance their half-life in the human body. PEGylated nanoparticles are widely used and have been developed into various types of nanomedicine. For example, PEG-modified liposomal doxorubicin (Caelyx and Lipo-Dox) has been used to treat ovarian, breast carcinomas and Kaposi's sarcoma 1,2. PEGylated Interferon (Pegasys 3,4 , PEG-Intron 5) was employed as a long-term therapeutic agent for hepatitis C. Several PEGylated polymeric micelle formulations, such as Paclitaxel and Cisplatin, are currently in phase I/II clinical trials for treatment of stomach cancer and solid tumors 6,7. PEG-modified imaging nanoparticles, such as quantum dots (QD) 8 and clinically approved super-paramagnetic iron oxide (PEG-SPIO) 9 have also been used to track the localization of tumors by optical
PLOS ONE, 2015
The development of effective adjuvant is the key factor to boost the immunogenicity of tumor cell... more The development of effective adjuvant is the key factor to boost the immunogenicity of tumor cells as a tumor vaccine. In this study, we expressed membrane-bound granulocytemacrophage colony-stimulating factor (GM-CSF) and interleukin-18 (IL-18) as adjuvants in tumor cells to stimulate immune response. B7 transmembrane domain fused GM-CSF and IL-18 was successfully expressed in the cell membrane and stimulated mouse splenocyte proliferation. Co-expression of GM-CSF and IL-18 reduced tumorigenesis (P<0.05) and enhanced tumor protective efficacy (P<0.05) significantly in comparison with GM-CSF alone. These results indicated that the combination of GM-CSF andIL-18 will enhance the immunogenicity of a cell-based anti-tumor vaccine. This membrane-bound approach can be applied to other cytokines for the development of novel vaccine strategies.
Veterinary journal (London, England : 1997), Jan 14, 2015
The envelope glycoprotein E2 of classical swine fever virus (CSFV) is widely used as a marker for... more The envelope glycoprotein E2 of classical swine fever virus (CSFV) is widely used as a marker for measuring vaccine efficacy and antibody titer. The glycosylation profile of E2 may affect the immunogenicity of the vaccine and the timing of re-vaccination. In this study, a human embryonic kidney cell line was used to secrete fully-glycosylated CSFV E2, which was then coated onto ELISA plates without purification or adjustment. The resulting E2-secreting medium-direct-coating (E2-mDc) ELISA was successfully used to measure anti-E2 antibody titers in vaccinated and field pig sera samples. Compared with a virus neutralization test (as standard), the E2-mDc ELISA was found to be more accurate (90%) than a commercial CSFV antibody diagnostic kit (62%). In conclusion, the mammalian cell-secreted antigen can provide cheap, accurate and effective assays for vaccine efficacy and disease diagnoses.
TheScientificWorldJournal, 2015
Glucuronidation is a major metabolism process of detoxification for carcinogens, 4-(methylnitrosa... more Glucuronidation is a major metabolism process of detoxification for carcinogens, 4-(methylnitrosamino)-1-(3-pyridy)-1-butanone (NNK) and 1,2-dimethylhydrazine (DMH), of reactive oxygen species (ROS). However, intestinal E. coli β-glucuronidase (eβG) has been considered pivotal to colorectal carcinogenesis. Specific inhibition of eβG may prevent reactivating the glucuronide-carcinogen and protect the intestine from ROS-mediated carcinogenesis. In order to develop specific eβG inhibitors, we found that 59 candidate compounds obtained from the initial virtual screening had high inhibition specificity against eβG but not human βG. In particular, we found that compounds 7145 and 4041 with naphthalenylidene-benzenesulfonamide (NYBS) are highly effective and selective to inhibit eβG activity. Compound 4041 (IC50 = 2.8 μM) shows a higher inhibiting ability than compound 7145 (IC50 = 31.6 μM) against eβG. Furthermore, the molecular docking analysis indicates that compound 4041 has two hy...
International journal of molecular sciences, 2015
Akt acts as a pivotal regulator in the PI3K/Akt signaling pathway and represents a potential drug... more Akt acts as a pivotal regulator in the PI3K/Akt signaling pathway and represents a potential drug target for cancer therapy. To search for new inhibitors of Akt kinase, we performed a structure-based virtual screening using the DOCK 4.0 program and the X-ray crystal structure of human Akt kinase. From the virtual screening, 48 compounds were selected and subjected to the Akt kinase inhibition assay. Twenty-six of the test compounds showed more potent inhibitory effects on Akt kinase than the reference compound, H-89. These 26 compounds were further evaluated for their cytotoxicity against HCT-116 human colon cancer cells and HEK-293 normal human embryonic kidney cells. Twelve compounds were found to display more potent or comparable cytotoxic activity compared to compound H-89 against HCT-116 colon cancer cells. The best results were obtained with Compounds a46 and a48 having IC50 values (for HCT-116) of 11.1 and 9.5 µM, respectively, and selectivity indices (IC50 for HEK-293/IC50 f...
Molecular cancer therapeutics, 2014
Beta-glucuronidase (βG) is a potential biomarker for cancer diagnosis and prodrug therapy. The ab... more Beta-glucuronidase (βG) is a potential biomarker for cancer diagnosis and prodrug therapy. The ability to image βG activity in patients would assist in personalized glucuronide prodrug cancer therapy. However, whole-body imaging of βG activity for medical usage is not yet available. Here, we developed a radioactive βG activity-based trapping probe for positron emission tomography (PET). We generated a (124)I-tyramine-conjugated difluoromethylphenol beta-glucuronide probe (TrapG) to form (124)I-TrapG that could be selectively activated by βG for subsequent attachment of (124)I-tyramine to nucleophilic moieties near βG-expressing sites. We estimated the specificity of a fluorescent FITC-TrapG, the cytotoxicity of tyramine-TrapG, and the serum half-life of (124)I-TrapG. βG targeting of (124)I-TrapG in vivo was examined by micro-PET. The biodistribution of (131)I-TrapG was investigated in different organs. Finally, we imaged the endogenous βG activity and assessed its correlation with t...
Biomaterials, 2014
Methoxy PEGylated nanoparticles (mPEG-NPs) are increasingly used for cancer imaging and therapy. ... more Methoxy PEGylated nanoparticles (mPEG-NPs) are increasingly used for cancer imaging and therapy. Here we describe a general and simple approach to confer tumor tropism to any mPEG-NP. We demonstrate this approach with humanized bispecific antibodies (BsAbs) that can bind to both mPEG molecules on mPEG-NPs and to EGFR or HER2 molecules overexpressed on the surface of cancer cells. Simple mixing of BsAbs with mPEG-NPs can mediate preferential binding of diverse mPEG-NPs to cancer cells that overexpress EGFR or HER2 under physiological conditions and significantly increase cancer cell killing by liposomal doxorubicin to EGFR þ and HER2 þ cancer cells. BsAbs modification also enhanced accumulation of fluorescence-labeled NPs and significantly increased the anticancer activity of drugloaded NPs to antigen-positive human tumors in a mouse model. Anti-mPEG BsAbs offer a simple one-step method to confer tumor specificity to mPEG-NPs for enhanced tumor accumulation and improved therapeutic efficacy.
Macromolecules, 2014
Quantitative pharmacokinetic analysis of methoxy-poly(ethylene glycol) (mPEG) and mPEGylated mole... more 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. α-mPEG sandwich ELISA could quantify the highermolecular-weight of mPEG (2, 5, and 20 kDa) and mPEGylated molecules. α-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, α-mPEG cell-based ELISA was unaffected by the presence of 10% human serum or murine serum. We further demonstrate that the α-mPEG cell-based ELISA determined similar pharmacokinetics of mPEG 5K as traditional gamma counting of 131 I-mPEG 5K. The α-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. ■ INTRODUCTION PEGylation is a FDA-approved technology for enhancing the bioavailability, safety, stability, and efficacy of a wide range of small molecules, proteins, liposomes, or nanoparticles. 1,2 The monomethoxylated form of PEG (mPEG) is widely used for clinical drug modification 3,4 because mPEG modification can reduce nonspecific binding and prevent recognition by the reticuloendothelial system (RES) in vivo. 5,6 Hence, a wide range
Bioconjugate Chemistry, 2013
Attachment of poly(ethylene glycol) to proteins can mask immune epitopes to increase serum half-l... more 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 antiinterferon 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 131 I-Pegasys in pharmacokinetic studies in mice. The anti-PEG sandwich ELISA provides a sensitive, accurate, and convenient quantitative measurement of PEGylated protein drugs.
PLoS ONE, 2014
Single-chain variable fragments (scFvs) serve as an alternative to full-length monoclonal antibod... more 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 (aEGFR) was fused with osmY (N-and C-termini) and periplasmic leader sequence (pelB) to generate aEGFR-osmY, osmY-aEGFR, and pelB-aEGFR (control), respectively. In comparison with the control, both the osmYfused aEGFR scFvs were soluble and secreted into the LB medium. Furthermore, the yield of soluble aEGFR-osmY was 20fold higher, and the amount of secreted protein was 250-fold higher than that of osmY-aEGFR. In addition, the antigenbinding activity of both the osmY-fused aEGFRs was 2-fold higher than that of the refolded pelB-aEGFR from inclusion bodies. Similar results were observed with aTAG72-osmY and aHer2-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 aEGFR protein.