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Activated fibroblasts are thought to play important roles in the progression of many solid tumors, but little is known about the mechanisms responsible for the recruitment of fibroblasts in tumors. Using several methods, we identified platelet-derived growth factor A (PDGFA) as the major fibroblast chemoattractant and mitogen from conditioned medium generated by the Calu-6 lung carcinoma cell line. In addition, we showed that Calu-6 tumors express significant levels of PDGFC, and that the levels of expression of these two PDGFRα ligands correlate strongly with the degree of stromal fibroblast infiltration into the tumor mass. The most intense expression of PDGFRα was observed in fibroblasts in the tumor outer rim. We subsequently showed that disrupting PDGFRα-mediated signaling results in significant inhibition of tumor growth in vivo. Furthermore, analysis of a compendium of microarray data revealed significant expression of PDGFA, PDGFC, and PDGFRα in human lung tumors. We propose that therapies targeting this stromal cell type may be effective in treating certain types of solid tumors.

PDF file - 234K, Baseline demographics for patients with and without VEGF-A samples in the AVF2107, E4599, AVAiL, and AVOREN trials, The GEN.038 VEGF-A ELISA, Standard curves for different VEGF-A isoforms in the GEN.038 assay, Correlation of baseline plasma VEGF-A levels with patient and tumor characteristics from AVOREN

Purpose: We evaluated the prognostic and predictive use of circulating VEGF-A levels in phase III trials of bevacizumab in colorectal cancer, lung cancer, and renal cell carcinoma.Methods: Baseline plasma samples from 1,816 patients were analyzed for VEGF-A using an ELISA, which recognizes the major isoforms with equivalent sensitivity. HR and 95% confidence intervals (CI) for study end points were estimated using Cox regression analysis. A subset of matched archival tumor samples was analyzed for VEGF-A expression using in situ hybridization.Results: Higher VEGF-A levels showed trends toward adverse prognostic significance in the control arms of multiple trials, reaching statistical significance for overall survival (OS) in AVF2107 (highest vs. lowest 50%: HR = 1.76; 95% CI, 1.28–2.41), AVAiL (HR = 1.52; 95% CI, 1.16–2.00), and AVOREN (HR = 1.67; 95% CI, 1.18–2.36). In predictive analyses, the HRs for progression-free survival were similar across low and high VEGF-A subgroups and favored bevacizumab-containing treatment. In the low VEGF-A subgroups, HRs (95% CIs) were 0.61 (0.43–0.87) in AVF2107, 0.71 (0.43–1.16) in E4599, 0.74 (0.59–0.94) in AVAiL (low-dose), 0.89 (0.70–1.13) in AVAiL (high-dose), and 0.56 (0.40–0.78) in AVOREN. Analyses of OS data have shown similar results. No correlation between primary tumor VEGF-A expression and plasma VEGF-A levels was observed.Conclusions: In this comprehensive evaluation, pretreatment total circulating VEGF-A was prognostic for outcome in metastatic colorectal, lung, and renal cell cancers, but it was not predictive for bevacizumab-based treatment benefit. Clin Cancer Res; 19(4); 929–37. ©2012 AACR.

Supplementary Figure 1 from Tumor-Driven Paracrine Platelet-Derived Growth Factor Receptor α Signaling Is a Key Determinant of Stromal Cell Recruitment in a Model of Human Lung Carcinoma

Supplementary Figure 6 from A Therapeutic Anti–VEGF Antibody with Increased Potency Independent of Pharmacokinetic Half-life

Supplementary Data 1 from Superior In vivo Efficacy of Afucosylated Trastuzumab in the Treatment of HER2-Amplified Breast Cancer

Supplementary Figure 2 from A Therapeutic Anti–VEGF Antibody with Increased Potency Independent of Pharmacokinetic Half-life

Supplementary Figure 1 from A Therapeutic Anti–VEGF Antibody with Increased Potency Independent of Pharmacokinetic Half-life

Supplementary Figure 4 from A Therapeutic Anti–VEGF Antibody with Increased Potency Independent of Pharmacokinetic Half-life

Supplementary Figure Legends 1-6 from A Therapeutic Anti–VEGF Antibody with Increased Potency Independent of Pharmacokinetic Half-life

The enhancement of immune effector functions has been proposed as a potential strategy for increasing the efficacy of therapeutic antibodies. Here, we show that removing fucose from trastuzumab (Herceptin) increased its binding to FcγRIIIa, enhanced antibody-dependent cell-mediated cytotoxicity, and more than doubled the median progression-free survival when compared with conventional trastuzumab in treating preclinical models of HER2-amplified breast cancer. Our results show that afucosylated trastuzumab has superior efficacy in treating in vivo models of HER2-amplified breast cancer and support the development of effector function–enhanced antibodies for solid tumor therapy. Cancer Res; 70(11); 4481–9. ©2010 AACR.

Supplementary Figure 3 from A Therapeutic Anti–VEGF Antibody with Increased Potency Independent of Pharmacokinetic Half-life

Supplementary Figure Legends 1-3 from Superior In vivo Efficacy of Afucosylated Trastuzumab in the Treatment of HER2-Amplified Breast Cancer

Supplementary Data 2 from Superior In vivo Efficacy of Afucosylated Trastuzumab in the Treatment of HER2-Amplified Breast Cancer

Supplementary Tables 1 - 4 from A Therapeutic Anti–VEGF Antibody with Increased Potency Independent of Pharmacokinetic Half-life

Supplementary Figure 5 from A Therapeutic Anti–VEGF Antibody with Increased Potency Independent of Pharmacokinetic Half-life

Diabetic macular edema (DME) is the leading cause of vision loss and blindness among working-age adults. Although current intravitreal anti-vascular endothelial growth factor (VEGF) therapies improve vision for many patients with DME, approximately half do not achieve the visual acuity required to drive. We therefore sought additional approaches to resolve edema and improve vision for these patients.We explored direct agonists of Tie2, a receptor known to stabilize vasculature and prevent leakage. We identified a multivalent PEG-Fab conjugate, Tie2.1-hexamer, that oligomerizes Tie2 and drives receptor activation and characterized its activities in vitro and in vivo.Tie2.1-hexamer normalized and stabilized intercellular junctions of stressed endothelial cell monolayers in vitro, suppressed vascular leak in mice under conditions where anti-VEGF alone was ineffective, and demonstrated extended ocular exposure and robust pharmacodynamic responses in non-human primates.Tie2.1-hexamer directly activates the Tie2 pathway, reduces vascular leak, and is persistent within the vitreal humor.Our study presents a promising potential therapeutic for the treatment of DME.

Transforming growth factor β (TGFβ) signaling has been recently shown to reduce antitumor response to PD-L1 blockade, leading to a renewed enthusiasm in developing anti-TGFβ therapies for potential combination with cancer immunotherapy agents. Inhibition of TGFβ signaling in nonclinical toxicology species is associated with serious adverse toxicities including cardiac valvulopathies and anemia. Previously, cardiovascular toxicities have been thought to be limited to small molecule inhibitors of TGFβ receptor and not considered to be a liability associated with pan-TGFβ neutralizing monoclonal antibodies (mAbs). Here, we report the toxicity findings associated with a potent pan-TGFβ neutralizing mAb (pan-TGFβ mAb; neutralizes TGFβ1, 2, and 3) after 5 weekly intravenous doses of 10, 30, and 100 mg/kg, followed by a 4-week recovery period, in mice and cynomolgus monkeys. Mortality was observed due to acute bleeding and cardiovascular toxicity in mice at ≥ 30 mg/kg and prolonged menstruation in female monkeys at 100 mg/kg. Additional findings considered to be on-target exaggerated pharmacology included generalized bleeding and cardiovascular toxicity in mice and monkeys; histopathologic changes in the teeth, tongue, and skin in mice; and abnormal wound healing and microscopic pathology in the bone in monkeys. Importantly, our data indicate that the cardiovascular toxicities associated with the inhibition of TGFβ signaling are not limited to small molecule inhibitors but are also observed following administration of a potent pan-TGFβ inhibiting mAb.

Purpose This study aims to address the following questions: What is enduring about consumer behavior on social media given that digital and social media (DSM) technologies change rapidly? Why do millennials use social media to the extent they do? The authors’ review revealed that a prevailing theoretical approach that may help answer these questions is inadequate. The technology acceptance model (TAM) from information systems was grafted into marketing to explain consumer technology adoption. TAM predicts Facebook adoption effectively, as demonstrated in the authors’ first study, but does not go beyond that in explaining the why’s behind its use. In a second study, the authors used the means-end approach (MEC) complementarily to unearth the why’s of millennials’ use of Facebook. Design/methodology/approach The authors used a mixed-methods design combining the structural modeling of TAM with the probing one-on-one interviews and laddering of MEC. Findings The authors found that the laddering process both widened and deepened TAM’s scope. It not only confirmed the importance of the TAM attributes, perceived ease of use and perceived usefulness, but it also revealed others, in determining adoption. It was also able to dig deeper from these to uncover a mesh of fundamental values that millennials used Facebook to satisfy, such as belongingness, pleasure, social acceptance and inner harmony, in their quest for inner and relational contentment. The authors also found negative aspects that kept consumers away, such as its lack of privacy and the overwhelming nature of unwanted video in its feed, tying these back to important values. Research limitations/implications The authors build on prior exploratory work relating to DSM use and uncover psychological drivers of consumer behavior on social media, by blending TAM in a consumer context, and the MEC approach. The TAM-MEC framework used here offers a technology-independent template for other DSM research, by focusing on how and why consumers use media socially. Practical implications Managerially, the authors discuss the building of sustainable marketing strategy on enduring consumer values rather than on transient attributes or technologies. The authors also discuss potential areas of vulnerability for Facebook, such as its increasing use of video and live content, which creates negative consumer sentiment and which may drive consumers to competitors. Originality/value By blending the quantitative TAM and the qualitative MEC, something that has not been done before in marketing, this research provides trustworthy answers to the research questions. In so doing, this study also contributes some cohesion to the fragmented DSM research field, as called for recently in prominent journals, by anchoring DSM study in well-established theories in marketing.

Assessing the risks associated with genotoxic compounds is challenging because of their complex genotoxicity and the difficulty in the dynamic monitoring of coexisting hazards. In this paper, DNA-assembly-based multistimulus responsive capsules that can detect multiple genotoxic agents simultaneously are presented. By exploiting the sequence- and reactivity-editable properties of DNA, DNA sequences in a DNA shell are designed to exhibit multivalent susceptibility against ultraviolet B radiation, aflatoxin B1, and styrene oxide. Upon exposure to genotoxicants, the developed DNA capsules dissociate because of the production of DNA adducts or aptamer-ligand complex-activated dehybridization, which results in the release of encapsulated fluorophores for a measure of the genotoxicant level. The fluorophore release kinetics for each genotoxicant is investigated. Moreover, the destruction behaviors of the developed capsules are evaluated in binary and ternary toxin mixtures. Multiple linear regression indicates the existence of a strong relationship between the fluorescent response and the genotoxicant level; the result highlights the significance of particular genotoxicant and the antagonistic effect of interacting genotoxic substances on capsule destruction. This DNA architecture allows the monitoring of human exposure to genotoxic agents, which enables the timely adoption of remedial measures, and benefits development of an endogenous genotoxin-responsive drug delivery system.

Transforming growth factor-β (TGFβ) is a key driver of fibrogenesis. Three TGFβ isoforms (TGFβ1, TGFβ2, and TGFβ3) in mammals have distinct functions in embryonic development; however, the postnatal pathological roles and activation mechanisms of TGFβ2 and TGFβ3 have not been well characterized. Here, we show that the latent forms of TGFβ2 and TGFβ3 can be activated by integrin-independent mechanisms and have lower activation thresholds compared to TGFβ1. Unlike TGFB1, TGFB2 and TGFB3 expression is increased in human lung and liver fibrotic tissues compared to healthy control tissues. Thus, TGFβ2 and TGFβ3 may play a pathological role in fibrosis. Inducible conditional knockout mice and anti-TGFβ isoform-selective antibodies demonstrated that TGFβ2 and TGFβ3 are independently involved in mouse fibrosis models in vivo, and selective TGFβ2 and TGFβ3 inhibition does not lead to the increased inflammation observed with pan-TGFβ isoform inhibition. A cocrystal structure of a TGFβ2-anti-TGFβ2/3 antibody complex reveals an allosteric isoform-selective inhibitory mechanism. Therefore, inhibiting TGFβ2 and/or TGFβ3 while sparing TGFβ1 may alleviate fibrosis without toxicity concerns associated with pan-TGFβ blockade.

A phage‐derived human monoclonal antibody against VEGF‐C was developed as a potential anti‐tumor therapeutic and exhibited fast clearance in preclinical species, with notably faster clearance in serum than in plasma. The purpose of this work was to understand the factors contributing to its fast clearance. In vitro incubations in animal and human blood, plasma, and serum were conducted with radiolabeled anti‐VEGF‐C to determine potential protein and cell‐based interactions with the antibody as well as any matrix‐dependent recovery dependent upon the matrix. A tissue distribution study was conducted in mice with and without heparin infusion in order to identify a tissue sink and determine whether heparin could affect antibody recovery from serum and/or plasma. Incubation of radiolabeled anti‐VEGF‐C in human and animal blood, plasma, or serum revealed that the antibody formed a complex with an endogenous protein, likely VEGF‐C. This complex was trapped within the blood clot during serum preparation from blood, but not within the blood cell pellet during plasma preparation. Low level heparin infusion in mice slowed down clot formation during serum preparation and allowed for better recovery of the radiolabeled antibody in serum. No tissue sink was found in mice. Thus, during this characterization, we determined that the blood sampling matrix greatly impacted the amount of antibody recovered in the samples, therefore, altering its derived pharmacokinetic parameters. Target biology should be considered when selecting appropriate sampling matrices for PK analysis.

Outer membrane proteins (OMPs) in Gram-negative bacteria dictate permeability of metabolites, antibiotics, and toxins. Elucidating the structure-function relationships governing OMPs within native membrane environments remains challenging. We constructed a diverse library of >3000 monoclonal antibodies to assess the roles of extracellular loops (ECLs) in LptD, an essential OMP that inserts lipopolysaccharide into the outer membrane of Escherichia coli. Epitope binning and mapping experiments with LptD-loop-deletion mutants demonstrated that 7 of the 13 ECLs are targeted by antibodies. Only ECLs inaccessible to antibodies were required for the structure or function of LptD. Our results suggest that antibody-accessible loops evolved to protect key extracellular regions of LptD, but are themselves dispensable. Supporting this hypothesis, no α-LptD antibody interfered with essential functions of LptD. Our experimental workflow enables structure-function studies of OMPs in native cellular environments, provides unexpected insight into LptD, and presents a method to assess the therapeutic potential of antibody targeting., eLife digest The overuse and misuse of antibiotics has led to the rise of multi-drug resistant bacteria which threaten global public health. Antibiotics interfere with essential processes in bacteria so they are unable to divide or survive, but over time, the microbes have found ways to become immune to the drugs. New antibiotics are now desperately needed. Gram-negative bacteria are wrapped in an outer membrane made of large molecules called lipopolysaccharides. This structure is an extra barrier to molecules (such as drugs) that try to enter the cell, but it could also hold new targets for antibiotics to exploit. A protein called LptD is embedded in the outer membrane, where it inserts new lipopolysaccharides. It is critical for bacteria to grow and survive, and is a relatively new potential target for antibiotic development. The protein has a number of ‘extracellular loops’ that extend into the environment, but their roles in the structure and the activity of LptD are still largely unknown. This is partly due to a lack of tools to investigate these elements. In response, Storek et al. built a library of over 3,000 custom antibodies, which are small Y-shaped proteins that can each recognise a specific portion in one of the extracellular loops and potentially incapacitate LptD. The antibodies were used to target LptD in its native environment, when it is embedded in the bacteria. In parallel, mutant bacteria were created in which the loops were genetically removed one by one to assess their importance for LptD activity. The experiments revealed that although the antibodies could target most extracellular loops, they could not target the few loops that were essential for LptD to work properly. This suggests that antibody-accessible loops are expendable and that these structures could serve to shield other regions of LptD which are critical for survival. The findings will help to prioritise research that develops other approaches to inhibit LptD. Finally, the antibody workflow designed by Storek et al. can serve as a road map to study other membrane proteins in their native cellular environment.

Purpose This research aims to examine how source of funds (paying with company’s funds versus personal funds) affects buyer’s judgments of price fairness and via these judgments, buyer’s response to prices. Design/methodology/approach A scenario-based experiment is used (N = 200). To test the hypotheses, the authors run moderated mediation regression analyses with the help of the PROCESS macro. Findings Drawing on fairness heuristics theory, the authors hypothesize and find that relative to when paying with personal funds, when paying with company’s funds, the perceived price difference plays a less significant role, whereas the perceived social acceptability of the pricing practice underlying the price difference plays a more important role in shaping price fairness judgments and, via these judgments, buyer’s response to prices. Practical implications The findings generate advice for companies that serve both the business and personal segments (e.g. airlines and hotels). Buyers in the personal segment typically pay with their own money. To persuade these buyers that a price is fair, it is crucial to show that the price represents a good deal for them. Buyers in the business segment often pay with company’s fund. Companies have more flexibility in charging different prices, but they should make sure that the reasons for the price difference are socially acceptable. Originality/value This research shows how the relative role of price difference versus social acceptability in price fairness judgments varies as a function of source of funds and how an inconsistency between price difference and its economic impact affects price fairness judgments.

Total online retail spending in China reached $427 billion in 2014 and is expected to surpass $1 trillion in 2018 (Chu and Wong 2015). In addition, the number of online stores in China has been rapidly increasing. The present study proposes a model based on the theoretical frameworks of technology readiness (Parasuraman et al. 2005) and e-service quality (Parasuraman and Malhotra 2005), and tested the model on consumers from China. The results indicate that consumer technology readiness positively influences the perceived efficiency, system availability, fulfillment, and privacy dimensions of e-service quality as it relates to the online retailing environment in China. Moreover, the influence of technology readiness on the intention to purchase online in the future is both direct and mediated by the dimensions of perceived e-service quality. Implications and future research suggestions are also discussed.

Antigen-binding fragments (Fab) and F(ab′)2 antibodies serve as alternative formats to full-length anti-bodies in therapeutic and immune assays. They provide the advantage of small size, short serum half-life, and lack of effector function. Several proteases associated with invasive diseases are known to cleave antibodies in the hinge-region, and this results in anti-hinge antibodies (AHA) toward the neoepitopes. The AHA can act as surrogate Fc and reintroduce the properties of the Fc that are otherwise lacking in antibody fragments. While this response is desired during the natural process of fighting disease, it is commonly unwanted for therapeutic antibody fragments. In our study, we identify a truncation in the lower hinge region of the antibody that maintains efficient proteolytic cleavage by IdeS protease. The resulting neoepitope at the F(ab′)2 C-terminus does not have detectable binding of pre-existing AHA, providing a practical route to produce F(ab′)2 in vitro by proteolytic digestion when the binding of pre-existing AHA is undesired. We extend our studies to the upper hinge region of the antibody and provide a detailed analysis of the contribution of C-terminal residues of the upper hinge of human IgG1, IgG2 and IgG4 to pre-existing AHA reactivity in human serum. While no pre-existing antibodies are observed toward the Fab of IgG2 and IgG4 isotype, a significant response is observed toward most residues of the upper hinge of human IgG1. We identify a T225L variant and the natural C-terminal D221 as solutions with minimal serum reactivity. Our work now enables the production of Fab and F(ab′)2 for therapeutic and diagnostic immune assays that have minimal reactivity toward pre-existing AHA.

For some antibodies intended for use as human therapeutics, reduced effector function is desired to avoid toxicities that might be associated with depletion of target cells. Since effector function(s), including antibody-dependent cell-mediated cytotoxicity (ADCC), require the Fc portion to be glycosylated, reduced ADCC activity antibodies can be obtained through aglycosylation of the human IgG1 isotype. An alternative is to switch to an IgG4 isotype in which the glycosylated antibody is known to have reduced effector function relative to glycosylated IgG1 antibody. ADCC activity of glycosylated IgG1 antibodies is sensitive to the fucosylation status of the Fc glycan, with both in vitro and in vivo ADCC activity increased upon fucose removal ("afucosylation"). The effect of afucosylation on activity of IgG4 antibodies is less well characterized, but it has been shown to increase the in vitro ADCC activity of an anti-CD20 antibody. Here, we show that both in vitro and in vivo activity of anti-CD20 IgG4 isotype antibodies is increased via afucosylation. Using blends of material made in Chinese hamster ovary (CHO) and Fut8KO-CHO cells, we show that ADCC activity of an IgG4 version of an anti-human CD20 antibody is directly proportional to the fucose content. In mice transgenic for human FcγRIIIa, afucosylation of an IgG4 anti-mouse CD20 antibody increases the B cell depletion activity to a level approaching that of the mIgG2a antibody.

IgG antibodies have been used to treat many diseases including cancer. IgG antibody-drug conjugates (ADCs) deliver cytotoxic drugs to target cells for cell elimination, but they have dose limiting toxicity due to target-independent uptake, including pinocytotic uptake. Neonatal Fc receptor (FcRn) recycles pinocytosed IgG in a pH-dependent manner and is the receptor responsible for the long half-life of IgG. Use of IgG variants with stronger FcRn binding at pH 6.0 for ADCs might improve recycling efficiency and reduce toxicity. However, these variants have residual FcRn binding at pH 7.4, which could lead to FcRn-mediated uptake and higher toxicity. Thus, the uptake of such variants at pH 7.4 needs to be evaluated. Here we report a reproducible and quantitative assay using an inducible HM7 colorectal cancer cell line to measure IgG uptake at endogenous and overexpressed FcRn levels. Our assay had comparable reproducibility at pH 6.0, 6.8 and 7.4. The wild type (WT) IgG had similar uptake at endogenous and overexpressed FcRn levels, as expected for pinocytotic uptake. We found similar uptake of a WT IgG and a stronger FcRn binding T307Q/N434A variant (QA variant) at endogenous FcRn levels at pH 7.4, although the QA variant had higher uptake at overexpressed FcRn levels. The QA variant also had higher uptake than the WT IgG at overexpressed FcRn levels at pH 6.8. Our assay can be used to characterize the stronger FcRn binding variants to aid in selection of suitable variants with low uptake at pH 7.4 for use as ADCs.

Outer membrane proteins (OMPs) in Gram-negative bacteria are essential for a number of cellular functions including nutrient transport and drug efflux. Escherichia coli BamA is an essential component of the OMP β-barrel assembly machinery and a potential novel antibacterial target that has been proposed to undergo large (~15 Å) conformational changes. Here, we explored methods to isolate anti-BamA monoclonal antibodies (mAbs) that might alter the function of this OMP and ultimately lead to bacterial growth inhibition. We first optimized traditional immunization approaches but failed to identify mAbs that altered cell growth after screening >3000 hybridomas. We then developed a “targeted boost-and-sort” strategy that combines bacterial cell immunizations, purified BamA protein boosts, and single hybridoma cell sorting using amphipol-reconstituted BamA antigen. This unique workflow improves the discovery efficiency of FACS + mAbs by >600-fold and enabled the identification of rare anti-BamA mAbs with bacterial growth inhibitory activity in the presence of a truncated lipopolysaccharide layer. These mAbs represent novel tools for dissecting the BamA-mediated mechanism of β-barrel folding and our workflow establishes a new template for the efficient discovery of novel mAbs against other highly dynamic membrane proteins.

Dissipating excess calories as heat through therapeutic stimulation of brown adipose tissues (BAT) has been proposed as a potential treatment for obesity-linked disorders. Here, we describe the generation of a humanized effector-less bispecific antibody that activates fibroblast growth factor receptor (FGFR) 1/βKlotho complex, a common receptor for FGF21 and FGF19. Using this molecule, we show that antibody-mediated activation of FGFR1/βKlotho complex in mice induces sustained energy expenditure in BAT, browning of white adipose tissue, weight loss, and improvements in obesity-associated metabolic derangements including insulin resistance, hyperglycemia, dyslipidemia and hepatosteatosis. In mice and cynomolgus monkeys, FGFR1/βKlotho activation increased serum high-molecular-weight adiponectin, which appears to contribute over time by enhancing the amplitude of the metabolic benefits. At the same time, insulin sensitization by FGFR1/βKlotho activation occurs even before the onset of weight loss in a manner that is independent of adiponectin. Together, selective activation of FGFR1/βKlotho complex with a long acting therapeutic antibody represents an attractive approach for the treatment of type 2 diabetes and other obesity-linked disorders through enhanced energy expenditure, insulin sensitization and induction of high-molecular-weight adiponectin., Highlights • A humanized bispecific antibody that selectively activates FGFR1/βKlotho complex was generated. • Anti-FGFR1/βKlotho agonist antibody induced sustained thermogenesis in brown fat and induced weight loss. • Anti-FGFR1/βKlotho agonist antibody improved insulin sensitivity even before the onset of weight loss.

Immuno-polymerase chain reaction (immuno-PCR) combines the specificity of antibodies with the amplification power of PCR to detect low levels of proteins. Here, we describe the development of a 384-well immuno-PCR method that uses streptavidin coated on a PCR plate to capture complexes of biotinylated capture antibody, antigen, and DNA-labeled detection antibody. Unbound molecules are removed by a wash step using a standard plate washer. Antibody-DNA molecules in bound complexes are then detected directly on the plate using real-time PCR. Circulating human vascular endothelial growth factor concentrations measured by this method correlated with measurements obtained from enzyme-linked immunosorbent assay (ELISA). Using this method, we developed an assay for human epidermal growth factor-like domain 7 (EGFL7), an extracellular matrix-bound angiogenic factor. EGFL7 is expressed at a higher level in certain cancers, although endogenous EGFL7 concentrations have not been reported. Our 384-well EGFL7 immuno-PCR assay can detect 0.51pM EGFL7 in plasma, approximately 16-fold more sensitive than the ELISA, utilizing the same antibodies. This assay detected EGFL7 in lysates of non-small-cell lung cancer and hepatocellular carcinoma cell lines and also hepatocellular carcinoma, breast cancer, and ovarian cancer tissues. This 384-well immuno-PCR method can be used to develop high-throughput biomarker assays.

Depletion of IgE-switched B cells may be an efficacious therapy for the treatment of allergic asthma. A human-ized monoclonal IgG1 antibody against the M1 prime segment of human membrane IgE on IgE-switched B cells can re-duce serum IgE by depleting IgE-producing cells. Here we report that removal of the core fucose in the Fc glycan of this anti-M1 prime antibody increases its binding affinity for human Fc RIII receptors compared to the wildtype antibody. The enhancement in binding to Fc RIII results in increased antibody potency for the in vitro depletion of IgE-positive cells in antibody-dependent cell-mediated cytotoxicity assays and for the in vivo reduction of serum IgE in mice reconstituted with human immune cells. This afucosylated anti-M1 prime antibody is known as quilizumab and is in clinical develop-ment for the treatment of allergic asthma. Keywords: ADCC, afucosylation, asthma, IgE, quilizumab. INTRODUCTION Approximately 5-10% of asthmatics have severe disease that is poorly controlled despite the use of standard combina-tion therapies consisting of high dose inhaled corticosteroids (ICS) with other controller medications, and there is a sig-nificant need for new therapies to treat these patients [1-3]. Targeting of the immunoglobulin E (IgE) using a humanized monoclonal antibody that binds and neutralizes free serum IgE results in a reduction in exacerbations, decreases in symptom scores and the use of rescue medications, and im-provements in quality of life in moderate-to-severe allergic asthmatics who remain symptomatic despite the use of high-dose ICS and long-acting

The purpose of this paper is to present the development and validation of a measurement model for student user satisfaction in a laptop environment. Using a “quasi Delphi” method in addition to contributions from prior research we used EFA and CFA (LISREL) to identify a five factor (14 item) measurement model that best fit the data. The structural model identified two factors related to overall student satisfaction. Suggestions and implications for how to use this measure are provided.

The antibody Fc region regulates antibody cytotoxic activities and serum half-life. In a therapeutic context, however, the cytotoxic effector function of an antibody is often not desirable and can create safety liabilities by activating native host immune defenses against cells expressing the receptor antigens. Several amino acid changes in the Fc region have been reported to silence or reduce the effector function of antibodies. These earlier studies focused primarily on the interaction of human antibodies with human Fc-γ receptors, and it remains largely unknown how such changes to Fc might translate to the context of a murine antibody. We demonstrate that the commonly used N297G (NG) and D265A, N297G (DANG) variants that are efficacious in attenuating effector function in primates retain potent complement activation capacity in mice, leading to safety liabilities in murine studies. In contrast, we found an L234A, L235A, P329G (LALA-PG) variant that eliminates complement binding and fixation as well as Fc-γ-dependent, antibody-dependent, cell-mediated cytotoxity in both murine IgG2a and human IgG1. These LALA-PG substitutions allow a more accurate translation of results generated with an "effectorless" antibody between mice and primates. Further, we show that both human and murine antibodies containing the LALA-PG variant have typical pharmacokinetics in rodents and retain thermostability, enabling efficient knobs-into-holes bispecific antibody production and a robust path to generating highly effector-attenuated bispecific antibodies for preclinical studies.