Development of NS2B-NS3 protease inhibitor that impairs Zika virus replication.

Zika virus (ZIKV) is a mosquito-borne flavivirus that causes severe neurological disorders, such as microcephaly in fetuses. Most recently, an outbreak of ZIKV started in Brazil in 2015. To date, no therapeutic agents have been approved to treat ZIKV infection in the clinic. Here, we screened a small molecule inhibitor that can inhibit the function of ZIKV non-structural protein 2B (NS2B)-NS3 protease (ZIKV NS2B-NS3 protease), thereby interfering with viral replication and spread. First, we identified the half maximal inhibitory concentration (IC50) of compound 3 (14.01 µM), 8 (6.85 µM), and 9 (14.2 µM) and confirmed that they are all non-competitive inhibitors. In addition, we have used the blind molecular docking method to simulate the inhibition area of three non-competitive inhibitors (compound 3, 8, and 9) with the ZIKV NS2B-NS3 protease. The results indicated that the four allosteric binding residues (Gln139, Trp148, Leu150, and Val220) could form hydrogen bonds or non-bonding interactions most frequently with the three compounds. The interaction might induce the reaction center conformation change of NS2B-NS3 protease to reduce catalyzed efficiency. The concentration of compounds required to reduce cell viability by 50% (CC50), and the concentration of compounds required to inhibit virus-induced cytopathic effect by 50% (EC50) of three potential compounds are >200 µM, 2.15 µM (compound 3), > 200 µM, 0.52 µM (compound 8) and 61.48 µM, 3.52 µM (compound 9), and Temoporfin are 61.05 µM, 2 µM, respectively. To select candidate compounds for further animal experiments, we analyzed the selectivity index (SI) of compound 3 (93.02), 8 (384.61), 9 (17.46), and Temoporfin (30.53, FDA-approved drug against cancer). Compound 8 has the highest SI value. Therefore, compound 8 was selected for verification in animal models. In vivo, compound 8 significantly delayed ZIKV-induced lethality and illness symptoms and decreased ZIKV-induced weight loss in a ZIKV-infected suckling mouse model. We conclude that compound 8 is worth further investigation for use as a potential future therapeutic agent against ZIKV infection.

Engineering a High-Affinity Anti-Methoxy Poly(ethylene glycol) (mPEG) Antibody for Sensitive Immunosensing of mPEGylated Therapeutics and mPEG Molecules.

Sensitive quantification of methoxy poly(ethylene glycol) (mPEG)-conjugated therapeutics for pharmacokinetic determination is critical for mPEGylated drug development. However, sensitive measurement of low-molecular-weight (lmw) mPEG compounds remains challenging due to epitope competition between backbone-specific anti-PEG antibodies. Here, we engineered a high-affinity methoxy-specific anti-mPEG antibody for sensitive quantification of free mPEG molecules and mPEGylated therapeutics. The affinity-enhanced h15-2Y antibody variant shows a 10.3-fold increase in mPEG-binding activity compared to parental h15-2b. h15-2Y-based sandwich ELISA can effectively quantify lmw mPEG5K and high-molecular-weight (hmw) mPEG20K at concentrations as low as 3.4 and 5.1 ng mL-1, respectively. Moreover, lmw mPEG compounds (560, 750, 1000, and 2000 Da) can be efficiently quantified via h15-2Y-based competitive ELISA with detection limits at nanomolar levels. This study provides a promising approach for application in the quantitative analysis of the various sizes of mPEG molecules to accelerate the timeline of mPEG-conjugated drug development.

Micro-PET imaging of hepatitis C virus NS3/4A protease activity using a protease-activatable retention probe.

Hepatitis C virus (HCV) NS3/4A protease is an attractive target for direct-acting antiviral agents. Real-time tracking of the NS3/4A protease distribution and activity is useful for clinical diagnosis and disease management. However, no approach has been developed that can systemically detect NS3/4A protease activity or distribution. We designed a protease-activatable retention probe for tracking HCV NS3/4A protease activity via positron emission topography (PET) imaging. A cell-penetrating probe was designed that consisted of a cell-penetrating Tat peptide, HCV NS3/4A protease substrate, and a hydrophilic domain. The probe was labeled by fluorescein isothiocyanate (FITC) and 124I in the hydrophilic domain to form a TAT-ΔNS3/4A-124I-FITC probe. Upon cleavage at NS3/4A substrate, the non-penetrating hydrophilic domain is released and accumulated in the cytoplasm allowing PET or optical imaging. The TAT-ΔNS3/4A-FITC probe selectively accumulated in NS3/4A-expressing HCC36 (NS3/4A-HCC36) cells/tumors and HCV-infected HCC36 cells. PET imaging showed that the TAT-ΔNS3/4A-124I-FITC probe selectively accumulated in the NS3/4A-HCC36 xenograft tumors and liver-implanted NS3/4A-HCC36 tumors, but not in the control HCC36 tumors. The TAT-ΔNS3/4A-124I-FITC probe can be used to represent NS3/4 protease activity and distribution via a clinical PET imaging system allowing. This strategy may be extended to detect any cellular protease activity for optimization the protease-based therapies.

Structural insights into Nirmatrelvir (PF-07321332)-3C-like SARS-CoV-2 protease complexation: a ligand Gaussian accelerated molecular dynamics study.

Coronavirus 3C-like protease (3CLpro) is found in SARS-CoV-2 virus, which causes COVID-19. 3CLpro controls virus replication and is a major target for target-based antiviral discovery. As reported by Pfizer, Nirmatrelvir (PF-07321332) is a competitive protein inhibitor and a clinical candidate for orally delivered medication. However, the binding mechanisms between Nirmatrelvir and 3CLpro complex structures remain unknown. This study incorporated ligand Gaussian accelerated molecular dynamics, the one-dimensional and two-dimensional potential of mean force, normal molecular dynamics, and Kramers' rate theory to determine the binding and dissociation rate constants (koff and kon) associated with the binding of the 3CLpro protein to the Nirmatrelvir inhibitor. The proposed approach addresses the challenges in designing small-molecule antiviral drugs.

A novel 2-phase residual U-net algorithm combined with optimal mass transportation for 3D brain tumor detection and segmentation.

Utilizing the optimal mass transportation (OMT) technique to convert an irregular 3D brain image into a cube, a required input format for a U-net algorithm, is a brand new idea for medical imaging research. We develop a cubic volume-measure-preserving OMT (V-OMT) model for the implementation of this conversion. The contrast-enhanced histogram equalization grayscale of fluid-attenuated inversion recovery (FLAIR) in a brain image creates the corresponding density function. We then propose an effective two-phase residual U-net algorithm combined with the V-OMT algorithm for training and validation. First, we use the residual U-net and V-OMT algorithms to precisely predict the whole tumor (WT) region. Second, we expand this predicted WT region with dilation and create a smooth function by convolving the step-like function associated with the WT region in the brain image with a [Formula: see text] blur tensor. Then, a new V-OMT algorithm with mesh refinement is constructed to allow the residual U-net algorithm to effectively train Net1-Net3 models. Finally, we propose ensemble voting postprocessing to validate the final labels of brain images. We randomly chose 1000 and 251 brain samples from the Brain Tumor Segmentation (BraTS) 2021 training dataset, which contains 1251 samples, for training and validation, respectively. The Dice scores of the WT, tumor core (TC) and enhanced tumor (ET) regions for validation computed by Net1-Net3 were 0.93705, 0.90617 and 0.87470, respectively. A significant improvement in brain tumor detection and segmentation with higher accuracy is achieved.

Inhibition of gut microbial ß-glucuronidase effectively prevents carcinogen-induced microbial dysbiosis and intestinal tumorigenesis.

The bidirectional interaction between carcinogens and gut microbiota that contributes to colorectal cancer is complicated. Reactivation of carcinogen metabolites by microbial ß-glucuronidase (ßG) 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 ßG inhibitor TCH-3511 in carcinogen azoxymethane (AOM)-treated APCMin/+ mice. AOM induced intestinal ßG activity, which was reflected in increases in the incidence, formation, and number of tumors in the intestine. Notably, inhibition of gut microbial ßG by TCH-3511 significantly reduced AOM-induced intestinal ßG 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 ßG in promoting AOM-induced gut microbial dysbiosis and intestinal tumorigenesis, indicating the chemoprotective benefit of gut microbial ßG inhibition against carcinogens via maintaining the gut microbiota balance and preventing cancer-associated gut microbial dysbiosis. Thus, the bacterial-specific ßG inhibitor TCH-3511 is a potential chemoprevention agent for colorectal cancer.

3D brain tumor segmentation using a two-stage optimal mass transport algorithm.

Optimal mass transport (OMT) theory, the goal of which is to move any irregular 3D object (i.e., the brain) without causing significant distortion, is used to preprocess brain tumor datasets for the first time in this paper. The first stage of a two-stage OMT (TSOMT) procedure transforms the brain into a unit solid ball. The second stage transforms the unit ball into a cube, as it is easier to apply a 3D convolutional neural network to rectangular coordinates. Small variations in the local mass-measure stretch ratio among all the brain tumor datasets confirm the robustness of the transform. Additionally, the distortion is kept at a minimum with a reasonable transport cost. The original [Formula: see text] dataset is thus reduced to a cube of [Formula: see text], which is a 76.6% reduction in the total number of voxels, without losing much detail. Three typical U-Nets are trained separately to predict the whole tumor (WT), tumor core (TC), and enhanced tumor (ET) from the cube. An impressive training accuracy of 0.9822 in the WT cube is achieved at 400 epochs. An inverse TSOMT method is applied to the predicted cube to obtain the brain results. The conversion loss from the TSOMT method to the inverse TSOMT method is found to be less than one percent. For training, good Dice scores (0.9781 for the WT, 0.9637 for the TC, and 0.9305 for the ET) can be obtained. Significant improvements in brain tumor detection and the segmentation accuracy are achieved. For testing, postprocessing (rotation) is added to the TSOMT, U-Net prediction, and inverse TSOMT methods for an accuracy improvement of one to two percent. It takes 200 seconds to complete the whole segmentation process on each new brain tumor dataset.

Selective activation of pro-anti-IL-1ß antibody enhances specificity for autoinflammatory disorder therapy.

Canakinumab is a fully human monoclonal antibody that specifically neutralizes human interleukin (IL)-1ß and has been approved by the US Food and Drug Administration for treating different types of autoinflammatory disorders such as cryopyrin-associated periodic syndrome, tumor necrosis factor receptor-associated periodic syndrome and systemic juvenile idiopathic arthritis. However, long-term systemic neutralization of IL-1ß by Canakinumab may cause severe adverse events such as serious upper respiratory tract infections and inflammation, thereby decreasing the quality of life of patients. Here, we used an IgG1 hinge as an Ab lock to cover the IL-1ß-binding site of Canakinumab by linking with matrix metalloprotease 9 (MMP-9) substrate to generate pro-Canakinumab that can be specifically activated in the inflamed regions in autoinflammatory diseases to enhance the selectivity and safety of treatment. The Ab lock significantly inhibited the IL-1ß-binding by 68-fold compared with Canakinumab, and MMP-9 completely restored the IL-1ß neutralizing ability of pro-Canakinumab within 60 min and blocked IL-1ß-downstream signaling and IL-1ß-regulated genes (i.e., IL-6). It is expected that MMP-9 cleavable and efficient Ab lock will be able to significantly enhance the selective reaction of Canakinumab at the disease site and reduce the on-target toxicities of Canakinumab during systemic circulation, thereby showing potential for development to improve the safety and quality of life of patients with autoinflammatory disorders in the future.

Enhancement of tumor tropism of mPEGylated nanoparticles by anti-mPEG bispecific antibody for ovarian cancer therapy.

Ovarian cancer is highly metastatic, with a high frequency of relapse, and is the most fatal gynecologic malignancy in women worldwide. It is important to elevate the drug susceptibility and cytotoxicity of ovarian cancer cells, thereby eliminating resident cancer cells for more effective therapeutic efficacy. Here, we developed a bispecific antibody (BsAb; mPEG × HER2) that can easily provide HER2+ tumor tropism to mPEGylated liposomal doxorubicin (PLD) and further increase the drug accumulation in cancer cells via receptor-mediated endocytosis, and improve the cytotoxicity and therapeutic efficacy of HER2+ ovarian tumors. The mPEG × HER2 can simultaneously bind to mPEG molecules on the surface of PLD and HER2 antigen on the surface of ovarian cancer cells. Simply mixing the mPEG × HER2 with PLD was able to confer HER2 specificity of PLD to HER2+ ovarian cancer cells and efficiently trigger endocytosis and enhance cytotoxicity by 5.4-fold as compared to non-targeted PLD. mPEG × HER2-modified PLD was able to significantly increase the targeting and accumulation of HER2+ ovarian tumor by 220% as compared with non-targeted PLD. It could also significantly improve the anti-tumor activity of PLD (P < 0.05) with minimal obvious toxicity in a tumor-bearing mouse model. We believe that the mPEG × HER2 can significantly improve the therapeutic efficacy, potentially reduce the relapse freqency and thereby achieve good prognosis in ovarian cancer patients.

Ab locks for improving the selectivity and safety of antibody drugs.

Monoclonal antibodies (mAbs) are a major targeted therapy for malignancies, infectious diseases, autoimmune diseases, transplant rejection and chronic inflammatory diseases due to their antigen specificity and longer half-life than conventional drugs. However, long-term systemic antigen neutralization by mAbs may cause severe adverse events. Improving the selectivity of mAbs to distinguish target antigens at the disease site from normal healthy tissue and reducing severe adverse events caused by the mechanisms-of-action of mAbs is still a pressing need. Development of pro-antibodies (pro-Abs) by installing a protease-cleavable Ab lock is a novel and advanced recombinant Ab-based strategy that efficiently masks the antigen binding ability of mAbs in the normal state and selectively "turns on" the mAb activity when the pro-Ab reaches the proteolytic protease-overexpressed diseased tissue. In this review, we discuss the design and advantages/disadvantages of different Ab lock strategies, focusing particularly on spatial-hindrance-based and affinity peptide-based approaches. We expect that the development of different masking strategies for mAbs will benefit the local reactivity of mAbs at the disease site, increase the therapeutic efficacy and safety of long-term treatment with mAbs in chronic diseases and even permit scientists to develop Ab drugs for formerly undruggable targets and satisfy the unmet medical needs of mAb therapy.

Bispecific antibody (HER2 × mPEG) enhances anti-cancer effects by precise targeting and accumulation of mPEGylated liposomes.

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 (αmPEG scFv) was fused to the C-terminus of an anti-HER2 (αHER2) antibody to generate a HER2 × mPEG BsAb that retained the original efficacy of a targeted antibody while actively attracting mPEG-liposomes to accumulate at tumor sites. HER2 ×mPEG BsAb can simultaneously bind to HER2-high expressing MCF7/HER2 tumor cells and mPEG molecules on mPEG-liposomal doxorubicin (Lipo-Dox). Pre-incubation of HER2 × 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 × 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/neo1 tumors. Importantly, HER2 × 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 × 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 HER2×mPEG 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 pre-treatment of HER2×mPEG 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.

Use of syngeneic cells expressing membrane-bound GM-CSF as an adjuvant to induce antibodies against native multi-pass transmembrane protein.

Membrane antigens (mAgs) are important targets for the development of antibody (Ab) drugs. However, native mAgs are not easily prepared, causing difficulties in acquiring functional Abs. In this study, we present a platform in which human mAgs were expressed in native form on cell adjuvants made with membrane-bound cytokines that were then used immunize syngeneic mice directly. The membrane-bound cytokines were used as immune stimulators to enhance specific Ab responses against the desired mAgs. Then, mAgs-expressing xenogeneic cells were used for Ab characterization to reduce non-specific binding. We established cell adjuvants by expressing membrane-bound cytokines (mIL-2, mIL-18, or mGM-CSF) on BALB/3T3 cells, which were effective in stimulating splenocyte proliferation in vitro. We then transiently expressed ecotropic viral integration site 2B (EVI2B) on the adjuvants and used them to directly immunize BALB/c mice. We found that 3T3/mGM-CSF cells stimulated higher specific anti-EVI2B Ab response in the immunized mice than the other cell adjuvants. A G-protein coupled receptor (GPCR), CXCR2, was then transiently expressed on 3T3/mGM-CSF cell adjuvant to immunize mice. The immune serum exhibited relatively higher binding to xenogeneic 293 A/CXCR2 cells than 293 A cells (~3.5-fold). Several hybridoma clones also exhibited selective binding to 293 A/CXCR2 cells. Therefore, the cell adjuvant could preserve the native conformation of mAgs and exhibit anti-mAg Ab stimulatory ability, providing a more convenient and effective method to generate functional Abs, thus possibly accelerating Ab drug development.

Specific activation of pro-Infliximab enhances selectivity and safety of rheumatoid arthritis therapy.

During rheumatoid arthritis (RA) treatment, long-term injection of antitumor necrosis factor α antibodies (anti-TNFα Abs) may induce on-target toxicities, including severe infections (tuberculosis [TB] or septic arthritis) and malignancy. Here, we used an immunoglobulin G1 (IgG1) hinge as an Ab lock to cover the TNFα-binding site of Infliximab by linking it with matrix metalloproteinase (MMP) -2/9 substrate to generate pro-Infliximab that can be specifically activated in the RA region to enhance the selectivity and safety of treatment. The Ab lock significantly inhibits the TNFα binding and reduces the anti-idiotypic (anti-Id) Ab binding to pro-Infliximab by 395-fold, 108-fold compared with Infliximab, respectively, and MMP-2/9 can completely restore the TNFα neutralizing ability of pro-Infliximab to block TNFα downstream signaling. Pro-Infliximab was only selectively activated in the disease site (mouse paws) and presented similar pharmacokinetics (PKs) and bio-distribution to Infliximab. Furthermore, pro-Infliximab not only provided equivalent therapeutic efficacy to Infliximab but also maintained mouse immunity against Listeria infection in the RA mouse model, leading to a significantly higher survival rate (71%) than that of the Infliximab treatment group (0%). The high-selectivity pro-Infliximab maintains host immunity and keeps the original therapeutic efficiency, providing a novel strategy for RA therapy.

Enhanced drug internalization and therapeutic efficacy of PEGylated nanoparticles by one-step formulation with anti-mPEG bispecific antibody in intrinsic drug-resistant breast cancer.

For those patients with HER2-overexpressing breast cancer, treatment with PEGylated liposomal doxorubicin (PLD) is inefficacious due to the intrinsic low sensitivity to doxorubicin. A very large increase in drug accumulation by active targeting may enhance the therapeutic efficacy of PLD. We established a humanized bispecific antibody (BsAb; mPEG × HER2) which has dual specificity for methoxy-polyethylene glycol (mPEG) and human epidermal growth factor receptor 2 (HER2) to enhance the specificity, internalization and anticancer activity of PLD for cancer cells that overexpress HER2. One-step formulation of PLD with mPEG × HER2 converted the PLD into HER2 targeted liposomes that were stable at 4 °C in PBS as well as at 37 °C in the presence of serum. αHER2/PLD induced receptor-mediated endocytosis and enhanced doxorubicin accumulation in MCF7/HER2 (HER2-amplified) breast cancer cells. αHER2/PLD also displayed more than 200-fold increased cytotoxicity to MCF7/HER2 cells and 28-fold increased cytotoxicity to drug-resistant MDA-MB-361 cells with a physical deletion of the TOP2A gene. αHER2/PLD specifically accumulated doxorubicin in the nucleus of cancer cells in tumor-bearing mice and produced significantly greater antitumor activity against MCF7/HER2 (P < 0.0001) and MDA-MB-361 (P < 0.05) tumors as compared to untargeted PLD. Furthermore, the cardiotoxicity of αHER2/PLD was similar to that of PLD in human cardiomyocytes and in mice. Our results indicate that the one-step formulation of PLD by mPEG × HER2 is a simple method to confer tumor specificity, increase drug internalization and enhance the anticancer activity of PLD against HER2-overexpressing and doxorubicin-resistant breast cancer.

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.

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.

Endothelial angiogenesis is directed by RUNX1T1-regulated VEGFA, BMP4 and TGF-ß2 expression.

Tissue angiogenesis is intimately regulated during embryogenesis and postnatal development. Defected angiogenesis contributes to aberrant development and is the main complication associated with ischemia-related diseases. We previously identified the increased expression of RUNX1T1 in umbilical cord blood-derived endothelial colony-forming cells (ECFCs) by gene expression microarray. However, the biological relevance of RUNX1T1 in endothelial lineage is not defined clearly. Here, we demonstrate RUNX1T1 regulates the survival, motility and tube forming capability of ECFCs and EA.hy926 endothelial cells by loss-and gain-of function assays, respectively. Second, embryonic vasculatures and quantity of bone marrow-derived angiogenic progenitors are found to be reduced in the established Runx1t1 heterozygous knockout mice. Finally, a central RUNX1T1-regulated signature is uncovered and VEGFA, BMP4 as well as TGF-ß2 are demonstrated to mediate RUNX1T1-orchested angiogenic activities. Taken together, our results reveal that RUNX1T1 serves as a common angiogenic driver for vaculogenesis and functionality of endothelial lineage cells. Therefore, the discovery and application of pharmaceutical activators for RUNX1T1 will improve therapeutic efficacy toward ischemia by promoting neovascularization.

Enhancement Effect of a Variable Topology of a Membrane-Tethered Anti-Poly(ethylene glycol) Antibody on the Sensitivity for Quantifying PEG and PEGylated Molecules.

Sensitive quantification of the pharmacokinetics of poly(ethylene glycol) (PEG) and PEGylated molecules is critical for PEGylated drug development. Here, we developed a sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for PEG by tethering an anti-PEG antibody (AGP3) via tethers with different dimensions on the surface of 293T cells (293T/S-αPEG, short-type cells; 293T/L-αPEG, long-type cells; 293T/SL-αPEG, hybrid-type cells) to improve the binding capacity and detection limit for free PEG and PEGylated molecules. The binding capacity of hybrid-type cells for PEG-like molecules (CH3-PEG5K-FITC (FITC = fluorescein isothiocyanate) and eight-arm PEG20K-FITC) was at least 10-80-fold greater than that of 293T cells expressing anti-PEG antibodies with uniform tether lengths. The detection limit of free PEG (OH-PEG3K-NH2 and CH3-PEG5K-NH2) and PEG-like molecule (CH3-PEG5K-FITC, CH3-PEG5K-SHPP, and CH3-PEG5K-NIR797) was14-137 ng mL-1 in the hybrid-type cell-based sandwich ELISA. 293T/SL-αPEG cells also had significantly higher sensitivity for quantification of a PEGylated protein (PegIntron) and multiarm PEG macromolecules (eight-arm PEG20K-NH2 and eight-arm PEG40K-NH2) at 3.2, 16, and 16 ng mL-1, respectively. Additionally, the overall binding capacity of 293T/SL-αPEG cells for PEGylated macromolecules was higher than that of 293T/S-αPEG or 293T/L-αPEG cells. Anchoring anti-PEG antibodies on cells via variable-length tethers for cell-based sandwich ELISA, therefore, provides a sensitive, high-capacity method for quantifying free PEG and PEGylated molecules.

Proteomic analysis indicates the importance of TPM3 in esophageal squamous cell carcinoma invasion and metastasis.

Numerous esophageal squamous cell carcinoma (ESCC) patients exhibit tumor recurrence following radical resection. Invasion and metastasis are key factors in poor prognosis following esophagectomy. In the present study, two­dimensional gel electrophoresis (2-DE) and matrix­assisted laser desorption/ionization time-of-flight mass spectrometry were used to define patterns of protein expression in ESCC tissues at different pathological stages. The expression levels of identified proteins were determined by immunohistochemistry and western blotting. A total of fifteen protein spots with >2-fold differences were observed when comparing results of 2-DE for stage III and stage I ESCC tissue sample. A total of 12 proteins were identified by mass spectrometry analysis and database searches. The results of immunohistochemistry and western blotting demonstrated expression levels of tropomyosin 3 (TPM3) were higher in stage III ESCC tissue compared with stage I (P<0.05). The findings of the present study identified twelve proteins, which are closely associated with ESCC invasion and metastasis, apoptosis and cell signal transduction. Furthermore, the overexpression of TPM3 may be important in ESCC invasion and metastasis.