Pharmacological inhibition of bacterial ß-glucuronidase prevents irinotecan-induced diarrhea without impairing its antitumor efficacy in vivo.

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-38 G) during hepatic glucuronidation, and subsequently is exported into the intestine, where SN-38 G is hydrolyzed by bacterial ß-glucuronidase (ßG) to be SN-38, thus leading to intestinal toxicity. Thus, inhibition of the intestinal bacterial ßG 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 ßG (eßG)-specific inhibitor pyrazolo[4,3-c]quinoline derivative (TCH-3562) for the potential use in preventing CPT-11-induced diarrhea. TCH-3562 exhibited efficacious inhibitory potency of endogenous ßG activity in two anaerobes, Eubacteriumsp. and Peptostreptococcus anaerobius. Oral administration of TCH-3562 also effectively reduced the bacterial ßG 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, inhibition of the intestinal bacterial ßG activity by the specific inhibitor, TCH-3562, is promising to prevent CPT-11-induced diarrhea while maintaining its anti-tumor efficacy that may have clinical potentials for the treatment with CPT-11.

Pre-existing anti-polyethylene glycol antibody reduces the therapeutic efficacy and pharmacokinetics of PEGylated liposomes.

Rationale: Increasing frequency of human exposure to PEG-related products means that healthy people are likely to have pre-existing anti-PEG antibodies (pre-αPEG Ab). However, the influence of pre-αPEG Abs on the pharmacokinetics (PK) and therapeutic efficacy of LipoDox is unknown. Methods: We generated two pre-αPEG Ab mouse models. First, naïve mice were immunized with PEGylated protein to generate an endogenous αPEG Ab titer (endo αPEG). Second, monoclonal αPEG Abs were passively transferred (αPEG-PT) into naïve mice to establish a αPEG titer. The naïve, endo αPEG and αPEG-PT mice were intravenously injected with 111in-labeled LipoDox to evaluate its PK. Tumor-bearing naïve, endo αPEG and αPEG-PT mice were intravenously injected with 111in-labeled LipoDox to evaluate its biodistribution. The therapeutic efficacy of LipoDox was estimated in the tumor-bearing mice. Results: The areas under the curve (AUC)last of LipoDox in endo αPEG and αPEG-PT mice were 11.5- and 15.6- fold less, respectively, than that of the naïve group. The biodistribution results suggested that pre-αPEG Ab can significantly reduce tumor accumulation and accelerate blood clearance of 111In-labeled LipoDox from the spleen. The tumor volumes of the tumor-bearing endo αPEG and αPEG-PT mice after treatment with LipoDox were significantly increased as compared with that of the tumor-bearing naïve mice. Conclusions: Pre-αPEG Abs were found to dramatically alter the PK and reduce the tumor accumulation and therapeutic efficacy of LipoDox. Pre-αPEG may have potential as a marker to aid development of personalized therapy using LipoDox and achieve optimal therapeutic efficacy.

Specific Inhibition of Bacterial ß-Glucuronidase by Pyrazolo[4,3-c]quinoline Derivatives via a pH-Dependent Manner To Suppress Chemotherapy-Induced Intestinal Toxicity.

The direct inhibition of bacterial ß-glucuronidase (ßG) activity is expected to reduce the reactivation of glucuronide-conjugated 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 ßG-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 ßG 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 ßG-specific inhibitor that would allow this inhibitor to be used for the purpose of reducing drug toxicity.

Selective antibody activation through protease-activated pro-antibodies that mask binding sites with inhibitory domains.

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 pro-antibodies that attain high therapeutic potency yet reduced systemic on-target toxicity.

Real-time imaging of intestinal bacterial ß-glucuronidase activity by hydrolysis of a fluorescent probe.

Intestinal bacterial ß-glucuronidase (ßG) hydrolyzes glucuronidated metabolites to their toxic form in intestines, resulting in intestinal damage. The development of a method to inhibit ßG 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-ß-D-glucuronide (FDGlcU), to non-invasively image the intestinal bacterial ßG activity in nude mice. In vitro cell-based assays showed that the detection limit is 104 colony-forming units/well of ßG-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 ßG activity in intestines was detected 3 hours after gavage with FDGlcU. Following pretreatment with a bacterial ßG inhibitor, the fluorescence signal was significantly reduced in abdomens and excised intestines images. For a 4-day antibiotic treatment to deplete intestinal bacteria, the FDGlcU-based images showed that the ßG activity was decreased by 8.5-fold on day 4 and then gradually increased after treatment stopped. The results suggested that FDGlcU-based imaging revealed the in vitro and in vivo activity of intestinal bacterial ßG, which would facilitate pharmacodynamic studies of specific bacterial ßG inhibitors in animal studies.

Development of pH-Sensitive Cationic PEGylated Solid Lipid Nanoparticles for Selective Cancer-Targeted Therapy.

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 (IC50 = 0.37 ±0.21 ug/ml) or HCC36 cells (IC50 = 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.

Using anti-poly(ethylene glycol) bioparticles for the quantitation of PEGylated nanoparticles.

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 un-decomposed 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), PEG-stabilizing 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-111In) 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.

A Secondary Antibody-Detecting Molecular Weight Marker with Mouse and Rabbit IgG Fc Linear Epitopes for Western Blot Analysis.

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 anti-rabbit IgG secondary antibodies in standard immunoblots. Linear regression analysis of the M&R LE protein marker plotted as gel mobility versus the log of the marker molecular weights revealed good linearity with a correlation coefficient R2 value of 0.9965, indicating that the M&R LE protein marker displays high accuracy for determining protein molecular weights. This accurate, regular and auto-detected M&R LE protein marker may provide a simple, efficient and economical tool for protein analysis.

Optimization of an Anti-poly(ethylene glycol) (anti-PEG) Cell-Based Capture System To Quantify PEG and PEGylated Molecules.

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 dot quantification down to 7 pM. These results suggest that the combination of 293T/15-2b cells and 7A4 detection antibody is the optimal pair for sensitive quantification of free PEG, PEG-like molecules, and PEGylated proteins, whereas the 293T/3.3 cells combined with 15-2b are more suitable for quantifying PEGylated nanoparticles. The optimized anti-PEG cell-based sandwich ELISA can provide a sensitive, precise, and convenient tool for the quantification of a range of PEGylated molecules.

Development of Membrane-Bound GM-CSF and IL-18 as an Effective Tumor Vaccine.

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.

Direct coating of culture medium from cells secreting classical swine fever virus E2 antigen on ELISA plates for detection of E2-specific antibodies.

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.

Discovery of specific inhibitors for intestinal E. coli ß-glucuronidase through in silico virtual screening.

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 ß-glucuronidase (eßG) has been considered pivotal to colorectal carcinogenesis. Specific inhibition of eßG may prevent reactivating the glucuronide-carcinogen and protect the intestine from ROS-mediated carcinogenesis. In order to develop specific eßG inhibitors, we found that 59 candidate compounds obtained from the initial virtual screening had high inhibition specificity against eßG but not human ßG. In particular, we found that compounds 7145 and 4041 with naphthalenylidene-benzenesulfonamide (NYBS) are highly effective and selective to inhibit eßG activity. Compound 4041 (IC50 = 2.8 µM) shows a higher inhibiting ability than compound 7145 (IC50 = 31.6 µM) against eßG. Furthermore, the molecular docking analysis indicates that compound 4041 has two hydrophobic contacts to residues L361 and I363 in the bacterial loop, but 7145 has one contact to L361. Only compound 4041 can bind to key residue (E413) at active site of eßG via hydrogen-bonding interactions. These novel NYBS-based eßG specific inhibitors may provide as novel candidate compounds, which specifically inhibit eßG to reduce eßG-based carcinogenesis and intestinal injury.

Discovery of Akt kinase inhibitors through structure-based virtual screening and their evaluation as potential anticancer agents.

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 for HCT-116) of 12.5 and 16.1, respectively. Through structure-based virtual screening and biological evaluations, we have successfully identified several new Akt inhibitors that displayed cytotoxic activity against HCT-116 human colon cancer cells. Especially, Compounds a46 and a48 may serve as useful lead compounds for further development of new anticancer agents.

PET imaging of ß-glucuronidase activity by an activity-based 124I-trapping probe for the personalized glucuronide prodrug targeted therapy.

Beta-glucuronidase (ßG) is a potential biomarker for cancer diagnosis and prodrug therapy. The ability to image ßG activity in patients would assist in personalized glucuronide prodrug cancer therapy. However, whole-body imaging of ßG activity for medical usage is not yet available. Here, we developed a radioactive ßG 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 ßG for subsequent attachment of (124)I-tyramine to nucleophilic moieties near ßG-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. ßG 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 ßG activity and assessed its correlation with therapeutic efficacy of 9-aminocamptothecin glucuronide (9ACG) prodrug in native tumors. FITC-TrapG showed specific trapping at ßG-expressing CT26 (CT26/mßG) cells but not in CT26 cells. The native TrapG probe possessed low cytotoxicity. (124)I-TrapG preferentially accumulated in CT26/mßG but not CT26 cells. Meanwhile, micro-PET and whole-body autoradiography results demonstrated that (124)I-TrapG signals in CT26/mßG tumors were 141.4-fold greater than in CT26 tumors. Importantly, Colo205 xenografts in nude mice that express elevated endogenous ßG can be monitored by using infrared glucuronide trapping probes (NIR-TrapG) and suppressed by 9ACG prodrug treatment. (124)I-TrapG exhibited low cytotoxicity allowing long-term monitoring of ßG activity in vivo to aid in the optimization of prodrug targeted therapy.

One-step mixing with humanized anti-mPEG bispecific antibody enhances tumor accumulation and therapeutic efficacy of mPEGylated nanoparticles.

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(+) and HER2(+) cancer cells. BsAbs modification also enhanced accumulation of fluorescence-labeled NPs and significantly increased the anticancer activity of drug-loaded 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.

High-throughput sorting of the highest producing cell via a transiently protein-anchored system.

Developing a high-throughput method for the effecient selection of the highest producing cell is very important for the production of recombinant protein drugs. Here, we developed a novel transiently protein-anchored system coupled with fluorescence activated cell sorting (FACS) for the efficient selection of the highest producing cell. A furin cleavage peptide (RAKR) was used to join a human anti-epithelial growth factor antibody (αEGFR Ab) and the extracellular-transmembrane-cytosolic domains of the mouse B7-1 antigen (B7). The furin inhibitor can transiently switch secreted αEGFR Ab into a membrane-anchored form. After cell sorting, the level of membrane αEGFR Ab-RAKR-B7 is proportional to the amount of secreted αEGFR Ab in the medium. We further selected 23 αEGFR Ab expressing cells and demonstrated a high correlation (R2 = 0.9165) between the secretion level and surface expression levels of αEGFR Ab. These results suggested that the novel transiently protein-anchored system can easily and efficiently select the highest producing cells, reducing the cost for the production of biopharmaceuticals.

Functional production of a soluble and secreted single-chain antibody by a bacterial secretion system.

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 (αEGFR) was fused with osmY (N- and C-termini) and periplasmic leader sequence (pelB) to generate αEGFR-osmY, osmY-αEGFR, and pelB-αEGFR (control), respectively. In comparison with the control, both the osmY-fused αEGFR scFvs were soluble and secreted into the LB medium. Furthermore, the yield of soluble αEGFR-osmY was 20-fold higher, and the amount of secreted protein was 250-fold higher than that of osmY-αEGFR. In addition, the antigen-binding activity of both the osmY-fused αEGFRs was 2-fold higher than that of the refolded pelB-αEGFR from inclusion bodies. Similar results were observed with αTAG72-osmY and αHer2-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 αEGFR protein.

Sensitivity of PEGylated interferon detection by anti-polyethylene glycol (PEG) antibodies depends on PEG length.

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 131I-Pegasys in pharmacokinetic studies in mice. The anti-PEG sandwich ELISA provides a sensitive, accurate, and convenient quantitative measurement of PEGylated protein drugs.

A regularly spaced and self-revealing protein ladder for anti-tag Western blot analysis.

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.

An activity-based near-infrared glucuronide trapping probe for imaging ß-glucuronidase expression in deep tissues.

ß-glucuronidase is an attractive reporter and prodrug-converting enzyme. The development of near-IR (NIR) probes for imaging of ß-glucuronidase activity would be ideal to allow estimation of reporter expression and for personalized glucuronide prodrug cancer therapy in preclinical studies. However, NIR glucuronide probes are not yet available. In this work, we developed two fluorescent probes for detection of ß-glucuronidase activity, one for the NIR range (containing IR-820 dye) and the other for the visible range [containing fluorescein isothiocyanate (FITC)], by utilizing a difluoromethylphenol-glucuronide moiety (TrapG) to trap the fluorochromes in the vicinity of the active enzyme. ß-glucuronidase-mediated hydrolysis of the glucuronyl bond of TrapG generates a highly reactive alkylating group that facilitates the attachment of the fluorochrome to nucleophilic moieties located near ß-glucuronidase-expressing sites. FITC-TrapG was selectively trapped on purified ß-glucuronidase or ß-glucuronidase-expressing CT26 cells (CT26/mßG) but not on bovine serum albumin or non-ß-glucuronidase-expressing CT26 cells used as controls. ß-glucuronidase-activated FITC-TrapG did not interfere with ß-glucuronidase activity and could label bystander proteins near ß-glucuronidase. Both FITC-TrapG and NIR-TrapG specifically imaged subcutaneous CT26/mßG tumors, but only NIR-TrapG could image CT26/mßG tumors transplanted deep in the liver. Thus NIR-TrapG may provide a valuable tool for visualizing ß-glucuronidase activity in vivo.