The mechanism of action and biodistribution of a novel EGFR/VEGF bispecific fusion protein that exhibited superior antitumor activities.

Despite the promising clinical benefits of therapies targeting epidermal growth factor receptor (EGFR) or vascular endothelial growth factor (VEGF) with antibodies in various cancers, resistance to these therapies will inevitably develop following treatment. Recent studies suggest that crosstalk between the EGFR and VEGF signaling pathways might be involved in the development of resistance. Therefore, simultaneous blockade of EGFR and VEGF signaling may be able to counteract this resistance and improve clinical outcomes. Here, we devised a fusion protein with two copies of VEGFR1 domain 2 connected to the C-terminus of cetuximab that can simultaneously bind to EGFR and VEGF and effectively inhibit target cell growth mediated by these two pathways. Furthermore, the fusion protein could bring soluble VEGF into target cells for degradation through internalization upon binding to EGFR. Tissue distribution in mice confirmed that the fusion protein effectively accumulated in tumors compared to its mAb counterpart cetuximab. These features resulted in stronger antitumor efficacies in vivo than the combination of bevacizumab and cetuximab. Thus, we provide a promising new strategy for the treatment of EGFR-overexpressing cancers.

A strategy for the efficient construction of anti-PD1-based bispecific antibodies with desired IgG-like properties.

Targeting PD1/PDL1 with blocking antibodies for cancer therapy has shown promising benefits in the clinic, but only approximately 20-30% of patients develop durable clinical responses to the treatment. Bispecific antibodies (BsAbs) that combine PD1/PDL1 blockade with the modulation of another immune checkpoint target may have greater potential to enhance immune checkpoint blockade therapy. In this study, we identified an anti-PD1 monoclonal antibody, 609A, whose heavy chain can pair with a variety of light chains from different antibodies while maintaining its PD1 binding/blocking activity. Taking advantage of this property and using a linear F(ab')2 format, we successfully produced a series of tetravalent IgG-like BsAbs that simultaneously target PD1 and other immune checkpoint targets, including PDL1 and CTLA4. The BsAbs exhibited superior bioactivities in vitro and in vivo compared to their respective parental mAbs. Importantly, the BsAbs demonstrated the desired IgG-like physicochemical properties in terms of high-level expression, ease of purification to homogeneity, good stability and in vivo pharmacokinetics. In summary, we describe a novel and flexible plug-and-play platform to engineer IgG-like BsAbs with excellent development potential for clinical applications.

Bispecific antibody simultaneously targeting PD1 and HER2 inhibits tumor growth via direct tumor cell killing in combination with PD1/PDL1 blockade and HER2 inhibition.

Immune checkpoint blockade has shown significant clinical benefit in multiple cancer indications, but many patients are either refractory or become resistant to the treatment over time. HER2/neu oncogene overexpressed in invasive breast cancer patients associates with more aggressive diseases and poor prognosis. Anti-HER2 mAbs, such as trastuzumab, are currently the standard of care for HER2-overexpressing cancers, but the response rates are below 30% and patients generally suffer relapse within a year. In this study we developed a bispecific antibody (BsAb) simultaneously targeting both PD1 and HER2 in an attempt to combine HER2-targeted therapy with immune checkpoint blockade for treating HER2-positive solid tumors. The BsAb was constructed by fusing scFvs (anti-PD1) with the effector-functional Fc of an IgG (trastuzumab) via a flexible peptide linker. We showed that the BsAb bound to human HER2 and PD1 with high affinities (EC50 values were 0.2 and 0.14 nM, respectively), and exhibited potent antitumor activities in vitro and in vivo. Furthermore, we demonstrated that the BsAb exhibited both HER2 and PD1 blockade activities and was effective in killing HER2-positive tumor cells via antibody-dependent cellular cytotoxicity. In addition, the BsAb could crosslink HER2-positive tumor cells with T cells to form PD1 immunological synapses that directed tumor cell killing without the need of antigen presentation. Thus, the BsAb is a new promising approach for treating late-stage metastatic HER2-positive cancers.

Tumor-targeting anti-EGFR x anti-PD1 bispecific antibody inhibits EGFR-overexpressing tumor growth by combining EGFR blockade and immune activation with direct tumor cell killing.

We developed a strategy to combine conventional targeted therapy with immune checkpoint blockade using a tumor-targeting bispecific antibody (BsAb) to treat solid tumors. The BsAb was designed to simultaneously engage a tumor-associated antigen, epidermal growth factor receptor (EGFR), and programed cell death protein 1 (PD1). In addition to its direct anti-tumor activity via EGFR inhibition, the BsAb mediated efficient antibody-dependent cellular cytotoxicity (ADCC) and activated T cell antitumor im munity through blockade of PD1 from interacting with its counterpart, programed cell death ligand 1 (PDL1). Further, the BsAb exhibited a potent direct tumor cell killing activity in the presence of PBMC, most likely, via activating and, at the same time, physically engaging T cells with tumor cells. Taken together, we here illustrate a new strategy in the design and production of novel BsAbs with enhanced therapeutic efficacy through both direct tumor growth inhibition and T cell activation via tumor-targeted immune checkpoint blockade.

A new anti-HER2 antibody that enhances the anti-tumor efficacy of trastuzumab and pertuzumab with a distinct mechanism of action.

The majority of patients with metastatic breast cancer who are treated with the anti-HER2 monoclonal antibody, trastuzumab, generally develop resistance to the drug within a year after initiation of the treatment. Here we describe a new anti-HER2 humanized monoclonal antibody, 19H6-Hu, which binds to HER2 extracellular domain (ECD) with high affinity and inhibits proliferation of multiple HER2-overexpressing cancer cell lines as a single agent or in combination with trastuzumab. 19H6-Hu binds to the domain III in proximity to the domain IV of HER2 ECD, which differs from trastuzumab and pertuzumab. 19H6-Hu in combination with trastuzumab was more effective at blocking phosphorylation of ERK1/2, AKT(S473)and HER2 (Y1248) in HER2-positive cancer cells compared to trastuzumab alone or in combination with pertuzumab. Combination of three antibodies, 19H6-Hu, inetetamab (a trastuzumab analog) and pertuzumab exhibited much stronger inhibition of large NCI-N87 tumor xenografts (>400mm3) than the current standard of care, inetetamab (trastuzumab) plus Docetaxel (DTX), as well as the combination of 19H6-Hu, inetetamab and DTX. Our results highlight the functional variability of HER2 domains and provide a new insight into the design of HER2-targeting agents.

Important roles of CD32 in promoting suppression of IL-4 induced immune responses by a novel anti-IL-4Rα therapeutic antibody.

Asthma is characterized by airway hyperresponsiveness and inflammation, as well as underlying structural changes to the airways. Interleukin-4 (IL-4) is a key T-helper type 2 (Th2) cytokine that plays important roles in the pathogenesis of atopic and eosinophilic asthma. We developed a novel humanized anti-IL-4Rα antibody that can potently inhibit IL-4/IL-13-mediated TF-1 cell proliferation. Using monocytes isolated from human peripheral blood mononuclear cells (PBMCs), we revealed a critical role of CD32 in modulating the immune responses of monocytes in response to blockade of IL-4Rα signaling pathway. We, therefore, devised a new strategy to increase the efficacy of the anti-IL-4Rα monoclonal antibody for the treatment of asthma and other atopic diseases by co-engaging CD32 and IL-4Rα on monocytic cells by choosing IgG classes or Fc mutations with higher affinities for CD32. The antibody with selectively enhanced affinity for CD32A displayed superior suppression of IL-4-induced monocytes' activities, including the down-regulation of CD23 expression. Intriguingly, further analysis demonstrated that both CD32A and CD32B contributed to the enhancement of antibody-mediated suppression of CD23 expression from monocytes in response to blockade of IL-4Rα signaling. Furthermore, inhibition of IgE secretion from human PBMC by the antibody variants further suggests that the complex allergic inflammation mediated by IL-4/IL-4Rα signaling might result from a global network where multiple cell types that express multiple FcγRs are all involved, of which CD32, especially CD32A, is a key mediator. In this respect, our study provides new insights into designing therapeutic antibodies for targeting Th2 cytokine-mediated allergic pathogenesis.

Visible-Light Direct Conversion of Ethanol to 1,1-Diethoxyethane and Hydrogen over a Non-Precious Metal Photocatalyst.

Converting renewable biomass and their derivatives into chemicals and fuels has received much attention to reduce the dependence on fossil resources. Photocatalytic ethanol dehydrogenation-acetalization to prepare value-added 1,1-diethoxyethane and H2 was achieved over non-precious metal CdS/Ni-MoS2 catalyst under visible light. The system displays an excellent production rate and high selectivity of 1,1-diethoxyethane, 52.1 mmol g-1 h-1 and 99.2 %, respectively. In-situ electron spin resonance, photoluminescence spectroscopy and transient photocurrent responses were conducted to investigate the mechanism. This study provides a promising strategy for a green application of bioethanol.

Impairment of Akt activity by CYP2E1 mediated oxidative stress is involved in chronic ethanol-induced fatty liver.

Protein kinase B (PKB/Akt) plays important roles in the regulation of lipid homeostasis, and impairment of Akt activity has been demonstrated to be involved in the development of non-alcoholic fatty liver disease (NAFLD). Previous studies suggest that cytochrome P4502E1 (CYP2E1) plays causal roles in the pathogenesis of alcoholic fatty liver (AFL). We hypothesized that Akt activity might be impaired due to CYP2E1-induced oxidative stress in chronic ethanol-induced hepatic steatosis. In this study, we found that chronic ethanol-induced hepatic steatosis was accompanied with reduced phosphorylation of Akt at Thr308 in mice liver. Chronic ethanol exposure had no effects on the protein levels of phosphatidylinositol 3 kinase (PI3K) and phosphatase and tensin homologue deleted on chromosome ten (PTEN), and led to a slight decrease of phosphoinositide-dependent protein kinase 1 (PDK-1) protein level. Ethanol exposure resulted in increased levels of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE)-Akt adducts, which was significantly inhibited by chlormethiazole (CMZ), an efficient CYP2E1 inhibitor. Interestingly, N-acetyl-L-cysteine (NAC) significantly attenuated chronic ethanol-induced hepatic fat accumulation and the decline of Akt phosphorylation at Thr308. In the in vitro studies, Akt phosphorylation was suppressed in CYP2E1-expressing HepG2 (CYP2E1-HepG2) cells compared with the negative control HepG2 (NC-HepG2) cells, and 4-HNE treatment led to significant decrease of Akt phosphorylation at Thr308 in wild type HepG2 cells. Lastly, pharmacological activation of Akt by insulin-like growth factor-1 (IGF-1) significantly alleviated chronic ethanol-induced fatty liver in mice. Collectively, these results indicate that CYP2E1-induced oxidative stress may be responsible for ethanol-induced suppression of Akt phosphorylation and pharmacological modulation of Akt in liver may be an effective strategy for the treatment of ethanol-induced fatty liver.

Electrochemical oxygen reduction mechanism on FeN2-graphene.

Metal-coordinated nitrogen-doped carbons are highly active in promoting electrochemical oxygen reduction reactions (ORR). The detailed kinetic and thermodynamic ORR behavior on three different FeN2-graphene [FeN2-G (A), (B) and (C)] structures was investigated in this work. The results show that formation of these FeN2-G configurations is energetically favorable; however, not all of them are effective for ORR. The higher HOMO energy and smaller HOMO-LUMO gap of FeN2-G (A) and (C) make them have strong adsorption strengths to ORR intermediates, which leads to occupation the active sites on the catalysts during ORR, and thus loss of catalytic activity. Examination of the results of ∆G of each reduction step also drew the same conclusion. The ∆G of the elementary steps of the ORR at zero electrode potential vs. standard hydrogen electrode are downhill only on FeN2-G (B). Throughout the entire four-electron ORR, the reduction of O to OH displays the highest reaction barrier. When the potential is >0.19 V, the reduction of OH species into water is uphill. Therefore, ORR activity is limited by two rate-determining steps on FeN2-G (B) at high potential: O and OH reduction steps.

A site-holding effect of TiO2 surface hydroxyl in the photocatalytic direct synthesis of 1,1-diethoxyethane from ethanol.

To understand the mechanism of the photocatalytic direct synthesis of 1,1-diethoxyethane (DEE) from ethanol is vital for enhancing the reaction efficiency. Based on photocatalytic data of different phase TiO2 and F-TiO2 catalysts, radical trapping data, and GC-MS data, we proposed a photocatalytic mechanism for the preparation of both DEE in neat ethanol and 2,3-butanediol (2,3-BD) in ethanol-H2O using photocatalytic methods. In neat ethanol, hydroxyl isn't involved in the catalytic cyclic process but hydroxyl has an indirect site-holding effect, thus leading to more hydroxyl groups with higher activity. In ethanol-H2O, although the strong oxidant ˙OH radical is involved, fewer OH groups lead to higher selectivity of 2,3-BD. The interaction of the reactant/solvent with the surface group of the catalyst is important in the activity and selectivity of photocatalytic reactions. This finding gives fundamental insight into the role of TiO2 surface hydroxyl in the photocatalytic dehydrogenation process of alcohols and opens a promising path to obtaining both high selectivity and high conversion in TiO2-based photocatalytic activity.

Chlorine-Induced In Situ Regulation to Synthesize Graphene Frameworks with Large Specific Area for Excellent Supercapacitor Performance.

Three-dimensional (3D) graphene frameworks are usually limited by a complicated preparation process and a low specific surface area. This paper presents a facile suitable approach to effectively synthesize 3D graphene frameworks (GFs) with large specific surface area (up to 1018 m(2) g(-1)) through quick thermal decomposition from sodium chloroacetate, which are considerably larger than those of sodium acetate reported in our recent study. The chlorine element in sodium chloroacetate may possess a strong capability to induce in situ activation and regulate graphene formation during pyrolysis in one step. These GFs can be applied as excellent electrode materials for supercapacitors and can achieve an enhanced supercapacitor performance with a specific capacitance of 266 F g(-1) at a current density of 0.5 A g(-1).

Tunneling Interlayer for Efficient Transport of Charges in Metal Oxide Electrodes.

Due to the limited electronic conductivity, the application of many metal oxides that may have attractive (photo)-electrochemical properties has been limited. Regarding these issues, incorporating low-dimensional conducting scaffolds into the electrodes or supporting the metal oxides onto the conducting networks are common approaches. However, some key electronic processes like interfacial charge transfer are far from being consciously concerned. Here we use a carbon-TiO2 contact as a model system to demonstrate the electronic processes occurring at the metal-semiconductor interface. To minimize the energy dissipation for fast transfer of electrons from semiconductor to carbon scaffolds, facilitating electron tunneling while avoiding high energy-consuming thermionic emission is desired, according to our theoretical simulation of the voltammetric behaviors. To validate this, we manage to sandwich ultrathin TiO2 interlayers with heavy electronic doping between the carbon conductors and dopant-free TiO2. The radially graded distribution of the electronic doping along the cross-sectional direction of carbon conductor realized by immobilizing the dopant species on the carbon surface can minimize the energy consumption for contacts to both the carbon and the dopant-free TiO2. Our strategy provides an important requirement for metal oxide electrode design.

Ammonia-induced robust photocatalytic hydrogen evolution of graphitic carbon nitride.

We report a new and effective method to prepare high activity graphitic carbon nitride (g-C3N4) by a simple ammonia etching treatment. The obtained g-C3N4 displays a high BET surface area and enhanced electron/hole separation efficiency. The hydrogen evolution rates improved from 52 µmol h(-1) to 316.7 µmol h(-1) under visible light.

Multifunctional Nitrogen-Doped Carbon Nanodots for Photoluminescence, Sensor, and Visible-Light-Induced H2 Production.

Herein, multifunctional N-doped carbon nanodots (NCNDs) were prepared through the one-step hydrothermal treatment of yeast. Results show that the NCNDs can be used as a new photocatalyst to drive the water-splitting reaction under UV light. Moreover, the NCNDs can efficiently catalyze the hydrogen evolution reaction. Under visible-light irradiation, Eosin Y-sensitized NCNDs exhibit excellent activity for hydrogen evolution. The hydrogen evolution rate of NCNDs (without any modification and co-catalyst) reaches 107.1 µmol h(-1) (2142 µmol g(-1) h(-1) ). When Pt is loaded on the NCNDs, the hydrogen evolution rate reaches 491.2 µmol h(-1) (9824 µmol g(-1) h(-1)) under visible-light irradiation. In addition, the NCNDs show excellent fluorescent properties and can be applied as a fluorescent probe for the sensitive and selective detection of Fe(3+) .

A bi-functional antibody-receptor domain fusion protein simultaneously targeting IGF-IR and VEGF for degradation.

Bi-specific antibodies (BsAbs), which can simultaneously block 2 tumor targets, have emerged as promising therapeutic alternatives to combinations of individual monoclonal antibodies. Here, we describe the engineering and development of a novel, human bi-functional antibody-receptor domain fusion molecule with ligand capture (bi-AbCap) through the fusion of the domain 2 of human vascular endothelial growth factor receptor 1 (VEGFR1) to an antibody directed against insulin-like growth factor - type I receptor (IGF-IR). The bi-AbCap possesses excellent stability and developability, and is the result of minimal engineering. Beyond potent neutralizing activities against IGF-IR and VEGF, the bi-AbCap is capable of cross-linking VEGF to IGF-IR, leading to co-internalization and degradation of both targets by tumor cells. In multiple mouse xenograft tumor models, the bi-AbCap improves anti-tumor activity over individual monotherapies. More importantly, it exhibits superior inhibition of tumor growth, compared with the combination of anti-IGF-IR and anti-VEGF therapies, via powerful blockade of both direct tumor cell growth and tumor angiogenesis. The unique "capture-for-degradation" mechanism of the bi-AbCap is informative for the design of next-generation bi-functional anti-cancer therapies directed against independent signaling pathways. The bi-AbCap design represents an alternative approach to the creation of dual-targeting antibody fusion molecules by taking advantage of natural receptor-ligand interactions.

Understanding the formation mechanism of graphene frameworks synthesized by solvothermal and rapid pyrolytic processes based on an alcohol-sodium hydroxide system.

The determination of ways to facilitate the 2D-oriented assembly of carbons into graphene instead of other carbon structures while restraining the π-π stacking interaction is a challenge for the controllable bulk synthesis of graphene, which is vital both scientifically and technically. In this study, graphene frameworks (GFs) are synthesized by solvothermal and rapid pyrolytic processes based on an alcohol-sodium hydroxide system. The evolution mechanism of GFs is investigated systematically. Under sodium catalysis, the abundant carbon atoms produced by the fast decomposition of solvothermal intermediate self-assembled to graphene. The existence of abundant ether bonds may be favorable for 3D graphene formation. More importantly, GFs were successfully obtained using acetic acid as the carbon source in the synthetic process, suggesting the reasonability of analyzing the formation mechanism. It is quite possible to determine more favorable routes to synthesize graphene under this cognition. The electrochemical energy storage capacity of GFs was also studied, which revealed a high supercapacitor performance with a specific capacitance of 310.7 F/g at the current density of 0.2 A/g.

Intramolecular hydrogen bonds quench photoluminescence and enhance photocatalytic activity of carbon nanodots.

Understanding the photoluminescence (PL) and photocatalytic properties of carbon nanodots (CNDs) induced by environmental factors such as pH through surface groups is significantly important to rationally tune the emission and photodriven catalysis of CNDs. Through adjusting the pH of an aqueous solution of CNDs, it was found that the PL of CNDs prepared by ultrasonic treatment of glucose is strongly quenched at pH 1 because of the formation of intramolecular hydrogen bonds among the oxygen-containing surface groups. The position of the strongest PL peak and its corresponding excitation wavelength strongly depend on the surface groups. The origins of the blue and green emissions of CNDs are closely related to the carboxyl and hydroxyl groups, respectively. The deprotonated COO(-) and CO(-) groups weaken the PL peak of the CNDs and shift it to the red. CNDs alone exhibit photocatalytic activity towards degradation of Rhodamine B at different pH values under UV irradiation. The photocatalytic activity of the CNDs is the highest at pH 1 because of the strong intramolecular hydrogen bonds formed among the oxygen-containing groups.

Photocatalytic direct conversion of ethanol to 1,1- diethoxyethane over noble-metal-loaded TiO2 nanotubes and nanorods.

As one of the most important biomass platform molecules, ethanol needs to have its product chain chemically extended to meet future demands in renewable fuels and chemicals. Additionally, chemical conversion of ethanol under mild and green conditions is still a major challenge. In this work, ethanol is directly converted into 1,1-diethoxyethane (DEE) and H2 under mild photocatalytic conditions over platinum-loaded TiO2 nanotubes and nanorods. The reaction follows a tandem dehydrogenation-acetalization mechanism, in which ethanol is first dehydrogenated into acetaldehyde and H(+) ion by photogenerated holes, and then acetalization between acetaldehyde and ethanol proceeds through promotion by H(+) ions formed in real time. Excess H(+) ions are simultaneously reduced into H2 by photogenerated electrons. This photocatalytic process has a very high reaction rate over nanosized tubular and rod-like TiO2 photocatalysts, reaching 157.7 mmol g(-1) h(-1) in relatively low photocatalyst feeding. More importantly, the reaction is highly selective, with a nearly stoichiometric conversion of reacted ethanol into DEE. This photocatalytic dehydrogenation CO coupling of ethanol is a new green approach to the direct efficient conversion of ethanol into DEE and provides a promising channel for sustainable bioethanol applications.

Self-assembly of gridlike zinc oxide lamellae for chemical-sensing applications.

Hexagonal gridlike ZnO lamellae (GZL) with uniform thickness are synthesized by the o-phthalic acid-assisted hydrothermal method. Here, a systematic study of the assembly behaviors of gridlike ZnO lamellae obtained by the synergistic effect of urea and o-phthalic acid is presented. The morphology evolution and formation mechanism of GZL are also discussed in detail. The as-synthesized samples were characterized by powder X-ray diffraction, scanning electron microscopy, and transmission electron microscopy and electron diffraction spectroscopy. The results show that 2D ZnO lamellae are composed of nanotablets, which are jointed to form grids. The length and width of ZnO lamellae are about 1000 and 500 nm, respectively, and the thickness is about 30 nm. GZL morphology evolves from hexagonal ZnO nanotablets to lamellae with grids. The gas-sensing properties indicate that sensors fabricated from different materials present very different responses to volatile organic compounds. A GZL-based sensor has good response to acetaldehyde, and the response and recovery times are 1 and 6 s, respectively, when it was exposed to 1 ppm of acetaldehyde gas. The possibility of tuning the gas-sensing properties by adjusting the morphology makes GZL a novel candidate for more effective detection of a toxic, volatile gas with low concentration.