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The clinical benefit of PD-1 blockade can be improved by combination with CTLA4 inhibition but is commensurate with significant immune-related adverse events suboptimally limiting the doses of anti-CTLA4 mAb that can be used. MEDI5752 is a monovalent bispecific antibody designed to suppress the PD-1 pathway and provide modulated CTLA4 inhibition favoring enhanced blockade on PD-1+ activated T cells. We show that MEDI5752 preferentially saturates CTLA4 on PD-1+ T cells versus PD-1− T cells, reducing the dose required to elicit IL2 secretion. Unlike conventional PD-1/CTLA4 mAbs, MEDI5752 leads to the rapid internalization and degradation of PD-1. Moreover, we show that MEDI5752 preferentially localizes and accumulates in tumors providing enhanced activity when compared with a combination of mAbs targeting PD-1 and CTLA4 in vivo. Following treatment with MEDI5752, robust partial responses were observed in two patients with advanced solid tumors. MEDI5752 represents a novel immunotherapy engineered to preferentially inhibit CTLA4 on PD-1+ T cells. Significance: The unique characteristics of MEDI5752 represent a novel immunotherapy engineered to direct CTLA4 inhibition to PD-1+ T cells with the potential for differentiated activity when compared with current conventional mAb combination strategies targeting PD-1 and CTLA4. This molecule therefore represents a step forward in the rational design of cancer immunotherapy. See related commentary by Burton and Tawbi, p. 1008. This article is highlighted in the In This Issue feature, p. 995

China is a major producer and consumer of green tea and possesses numerous high-quality tea cultivars, which can be used to make green tea. However, the green teas produced from these tea cultivars vary markedly in aroma. In this study, a sensory evaluation method and headspace solid-phase microextraction combined with GC-MS were employed to determine the primary substances that contribute to the aromas and key differences in the aroma components of green tea produced from 20 tea cultivars. The results revealed 57 major volatile components, among which aldehydes, alcohols, and esters were the most prevalent. For the volatile components, the linalool, nonanal, and geraniol contents were the highest, followed by dimethyl sulfide, methyl salicylate, heptanal, and caproicacidhexneylester. The volatile components varied considerably among the green tea samples, and some contained substances with a high content that gave off unique aromas. The correlation analysis revealed significant correlations between the sensory evaluation scores and contents of linalool, geraniol, methyl salicylate, cis-linalool oxide, 3-carene, phenethyl alcohol, limonene, (Z)-3-cis-3-hexenyl isovalerate, citral, (E)-s-ocimene, alcohols, esters, and alkenes, as well as between the sensory evaluation scores and total volatile component contents. Aromatic substances with the highest odor activity values were, in descending order, linalool, capraldehyde, dimethyl sulfide, s-ionone, geraniol, nonanal, heptanal, (E)-2-nonenal, and caproicacidhexneylester. These substances were the major contributors to aroma formation in the green tea samples. The principal component analysis and orthogonal projections to the latent structure-discriminant analysis suggested that the 20 tea cultivars could be grouped into 6 categories and that the high content of linalool, nonanal, and geraniol substantially contributed to the categorization of the tea cultivars. A key variable analysis revealed that 23 volatile components played a critical role in the categorization of the tea cultivars.

Selenium (Se) is an essential element for human health and an important nutrient for plant growth. Selenite is the main form of Se available to plants in acidic soils. Previous studies have shown that phosphate transporters (PTHs) participate in selenite uptake in plants. Research on the PHT gene family is therefore vital for production of Se-rich products. Here, 23 CsPHT genes were identified in the tea (Camellia sinensis) genome and renamed based on homology with AtPHT genes in Arabidopsis thaliana. The CsPHT genes were divided into four subfamilies: PHT1, PHT3, PHT4, and PHO, containing nine, three, six, and five genes, respectively. Phylogenetic analysis indicated that fewer duplication events occurred in tea plants than in A. thaliana, rice, apple, and poplar. Genes in the same subfamily tended to share similar gene structures, conserved motifs, and potential functions. CsPHT genes were differentially expressed in various tissues and in roots under different Se levels, suggesting key roles in selenite uptake, translocation, and homeostasis. The results illuminate the contributions of CsPHT genes to selenite supply in tea plants, and lay a foundation for follow-up studies on their potential functions in this plant species.

Due to the accumulation of experiences on treating disease, tea began to develop to pluralism, and not limited to Camellia sinensis. The leaf buds of Eurya alata Kobuski and Camellia cuspidate were used to make Qianlincha (QLC) and Qiandingcha (QDC), which have special taste, aromas, and health benefits. In our study, the biochemical and volatile components of QLC and QDC were systematically analyzed and compared with a normal green tea (GT, C. sinensis). The biochemical and volatile components in the three tea samples were remarkably different. Compared with those in GT, QLC and QDC exhibited higher content of flavonoids and remarkably lower content of amino acids, catechins, and caffeine. High levels of flavonoids may play a crucial role in taste, liquor color, and health function of QLC and QDC. Low levels of amino acids, catechins, and caffeine may impart mouth-drying or velvety-like astringent taste, umami and refreshing taste of QLC and QDC was not as good as that of GT. High levels of linalool, geraniol, nonanal, dimethyl sulfide, and cis-jasmone may impart a clean and strongly floral or fruity aroma characteristic of QLC. High levels of linalool, 3,7-dimethyl-6-octen-3-ol, (-)-terpinen-4-ol, and terpenes may impart a strongly floral aroma characteristic of QDC.

Fear memory is a pivotal biological function by which organisms can predict possible danger to avoid or reduce harm. However, dysregulation of fear memory processing may lead to pathological fear or anxiety and produce serious clinical symptoms, such as post-traumatic stress disorder (PTSD). Iron deficiency (ID) is reported to inhibit the initiation of fear memory. In our study, we found that ferroportin1 (FPN1), the only known cellular iron export protein in mammals, and ablation in neurons and astrocytes caused iron deficiency in the cortex and hippocampus. However, little is known about its role in the development of fear memory. Moreover, direct evidence of the role of FPN1, or the related molecular mechanisms of such a role, in balancing brain iron homeostasis, especially in neuronal cells, is lacking. Herein, we deleted Fpn1 in mouse neurons, using Nestin-cre transgenic mice, and explored the impact on neuronal iron recycling and brain iron homeostasis in the cortex and hippocampus. We investigated the response of the mice to contextual fear and found that formation of fear memory was impeded after neuronal FPN1 depletion. We also found that FPN1 ablation in neurons and astrocytes caused an atypical expression of iron metabolism-related proteins in these two regions: decreased expression of DMT1, Ft-H, and Ft-L, and increased TfR1 expression. In addition, the decreased FPN1 in brain microvascular endothelial cells (BMVECs) also shed light on the cause of the decreased iron delivery to the brain through the blood-brain barrier (BBB). Our research highlights the major role played by FPN1 in brain iron homeostasis and identifies a potential target for the treatment of PTSD.

Green tea made from high amino acid content (HAAC) tea cultivars with different leaf colors exhibits many similar characteristics, such as slight bitterness, a rich aroma, and a refreshing and velvety taste. To determine differences in the key constituents of five HAAC tea cultivars with various leaf colors, the cultivars quality constituents were systematically analyzed and compared with that of a normal green tea cultivar. High theanine (Thea) content and the synthesis of Thea precursors are extremely important determinants of the character of these HAAC tea cultivars. The levels of most catechins, carotenoids, and chlorophylls in the HAAC tea cultivars were significantly lower than those in the normal tea cultivar, as was that of caffeine. The present study suggests that the inhibition of catechins (particularly epicatechins; ECs), chlorophylls, carotenoids, and caffeine biosyntheses in HAAC tea cultivars directs the metabolic network toward amino acid biosynthesis, particularly Thea biosynthesis.

Clostridium difficile infection is the leading cause of hospital-acquired diarrhea and is mediated by the actions of two toxins, TcdA and TcdB. The toxins perturb host cell function through a multistep process of receptor binding, endocytosis, low pH–induced pore formation, and the translocation and delivery of an N-terminal glucosyltransferase domain that inactivates host GTPases. Infection studies with isogenic strains having defined toxin deletions have established TcdB as an important target for therapeutic development. Monoclonal antibodies that neutralize TcdB function have been shown to protect against C. difficile infection in animal models and reduce recurrence in humans. Here, we report the mechanism of TcdB neutralization by PA41, a humanized monoclonal antibody capable of neutralizing TcdB from a diverse array of C. difficile strains. Through a combination of structural, biochemical, and cell functional studies, involving X-ray crystallography and EM, we show that PA41 recognizes a single, highly conserved epitope on the TcdB glucosyltransferase domain and blocks productive translocation and delivery of the enzymatic cargo into the host cell. Our study reveals a unique mechanism of C. difficile toxin neutralization by a monoclonal antibody, which involves targeting a process that is conserved across the large clostridial glucosylating toxins. The PA41 antibody described here provides a valuable tool for dissecting the mechanism of toxin pore formation and translocation across the endosomal membrane.

Clostridium difficile is a clinically significant pathogen that causes mild-to-severe (and often recurrent) colon infections. Disease symptoms stem from the activities of two large, multidomain toxins known as TcdA and TcdB. The toxins can bind, enter, and perturb host cell function through a multistep mechanism of receptor binding, endocytosis, pore formation, autoproteolysis, and glucosyltransferase-mediated modification of host substrates. Monoclonal antibodies that neutralize toxin activity provide a survival benefit in preclinical animal models and prevent recurrent infections in human clinical trials. However, the molecular mechanisms involved in these neutralizing activities are unclear. To this end, we performed structural studies on a neutralizing monoclonal antibody, PA50, a humanized mAb with both potent and broad-spectrum neutralizing activity, in complex with TcdA. Electron microscopy imaging and multiangle light-scattering analysis revealed that PA50 binds multiple sites on the TcdA C-terminal combined repetitive oligopeptides (CROPs) domain. A crystal structure of two PA50 Fabs bound to a segment of the TcdA CROPs helped define a conserved epitope that is distinct from previously identified carbohydrate-binding sites. Binding of TcdA to the host cell surface was directly blocked by either PA50 mAb or Fab and suggested that receptor blockade is the mechanism by which PA50 neutralizes TcdA. These findings highlight the importance of the CROPs C terminus in cell-surface binding and a role for neutralizing antibodies in defining structural features critical to a pathogen's mechanism of action. We conclude that PA50 protects host cells by blocking the binding of TcdA to cell surfaces.

Complement-dependent cytotoxicity (CDC) is a potent effector mechanism, engaging both innate and adaptive immunity. Although strategies to improve the CDC activity of antibody therapeutics have primarily focused on enhancing the interaction between the antibody crystallizable fragment (Fc) and the first subcomponent of the C1 complement complex (C1q), the relative importance of intrinsic affinity and binding valency of an antibody to the target antigen is poorly understood. Here we show that antibody binding affinity to a cell surface target antigen evidently affects the extent and efficacy of antibody-mediated complement activation. We further report the fundamental role of antibody binding valency in the capacity to recruit C1q and regulate CDC. More specifically, an array of affinity-modulated variants and functionally monovalent bispecific derivatives of high-affinity anti-epidermal growth factor receptor (EGFR) and anti-human epidermal growth factor receptor 2 (HER2) therapeutic immunoglobulin Gs (IgGs), previously reported to be deficient in mediating complement activation, were tested for their ability to bind C1q by biolayer interferometry using antigen-loaded biosensors and to exert CDC against a panel of EGFR and HER2 tumor cells of various histological origins. Significantly, affinity-reduced variants or monovalent derivatives, but not their high-affinity bivalent IgG counterparts, induced near-complete cell cytotoxicity in tumor cell lines that had formerly been shown to be resistant to complement-mediated attack. Our findings suggest that monovalent target engagement may contribute to an optimal geometrical positioning of the antibody Fc to engage C1q and deploy the complement pathway.

Late embryogenesis abundant (LEA) proteins are widely known to be present in higher plants and are believed to play important functional roles in embryonic development and abiotic stress responses. However, there is a current lack of systematic analyses on the LEA protein gene family in tea plant. In this study, a total of 48 LEA genes were identified using Hidden Markov Model profiles in C. sinensis, and were classified into seven distinct groups based on their conserved domains and phylogenetic relationships. Genes in the CsLEA_2 group were found to be the most abundant. Gene expression analyses revealed that all the identified CsLEA genes were expressed in at least one tissue, and most had higher expression levels in the root or seed relative to other tested tissues. Nearly all the CsLEA genes were found to be involved in seed development, and thirty-nine might play an important role in tea seed maturation concurrent with dehydration. However, only sixteen CsLEA genes were involved in seed desiccation, and furthermore, most were suppressed. Additionally, forty-six CsLEA genes could be induced by at least one of the tested stress treatments, and they were especially sensitive to high temperature stress. Furthermore, it was found that eleven CsLEA genes were involved in tea plant in response to all tested abiotic stresses. Overall, this study provides new insights into the formation of CsLEA gene family members and improves our understanding on the potential roles of these genes in normal development processes and abiotic stress responses in tea plant, particularly during seed development and desiccation. These results are beneficial for future functional studies of CsLEA genes that will help preserve the recalcitrant tea seeds for a long time and genetically improve tea plant.

Tea plant (Camellia sinensis) has strong enrichment ability for selenium (Se). Selenite is the main form of Se absorbed and utilized by tea plant. However, the mechanism of selenite absorption and accumulation in tea plant is still unknown. In this study, RNA sequencing (RNA-seq) was used to perform transcriptomic analysis on the molecular mechanism of selenite absorption and accumulation in tea plant. 397.98 million high-quality reads were obtained and assembled into 168,212 unigenes, 89,605 of which were extensively annotated. There were 60,582 and 1,362 differentially expressed genes (DEGs) in roots and leaves, respectively. RNA-seq results were further validated by quantitative RT-PCR. Based on GO terms, the unigenes were mainly involved in cell, binding and metabolic process. KEGG pathway enrichment analysis showed that predominant pathways included ribosome and protein processing in endoplasmic reticulum. Further analysis revealed that sulfur metabolism, glutathione metabolism, selenocompound metabolism and plant hormone signal transduction responded to selenite in tea plant. Additionally, a large number of genes of higher expressions associated with phosphate transporters, sulfur assimilation, antioxidant enzymes, antioxidant substances and responses to ethylene and jasmonic acid were identified. Stress-related plant hormones might play a signaling role in promoting sulfate/selenite uptake and assimilation in tea plant. Moreover, some other Se accumulation mechanisms of tea plant were found. Our study provides a possibility for controlling Se accumulation in tea plant through bio-technologies and will be helpful for breeding new tea cultivars.

The tea plant (Camellia sinensis (L.) O. Kuntze) is an economically important woody perennial nonalcoholic health beverage crop. Tea seeds are categorized as recalcitrant and are sensitive to dehydration treatment. However, the molecular basis of this phenomenon has not been investigated. Thus, we analyzed the genome-wide expression profiles of three dehydration stages using RNA-Seq and digital gene expression (DGE) technologies. We performed de novo assembly and obtained a total of 91,925 nonredundant unigenes, of which 58,472 were extensively annotated. By a hierarchical clustering of differentially expressed genes (DEGs), we found that 8929 DEGs were downregulated and 5875 DEGs were upregulated during dehydration treatment. A series of genes related to ABA biosynthesis and signal transduction, transcription factor, antioxidant enzyme, LEA protein, and proline metabolism that have been reported to function in dehydration process were found to be downregulated. Additionally, the expression profiles of 12 selected genes related to tea seed dehydration treatment were confirmed by qRT-PCR analysis. To our knowledge, this is the first genome-wide study elucidating the possible molecular mechanisms of sensitivity of recalcitrant tea seeds to dehydration. The results obtained in this study contribute to the preservation of tea seeds as genetic resources and can also be used to explore the mechanism of dehydration sensitivity of other recalcitrant seeds.

We describe herein a method to enable high throughput (HTP) screening of libraries of soluble proteins such as phage-derived clones of IgG, scFv-Fc, or other Fc-fusion proteins expressed in mammalian cells via adenovirus transduction. DNA fragments of antibody single chains (scFvs) and fragment antigen-binding (Fabs) from the positive clones of the third round of bacteriophage panning against a target antigen were batch reformatted into scFv-Fc or IgG in an oriP bearing entry vector and then recombined to an adenovirus vector through Gateway technology. The resulting antibody gene-containing adenovirus libraries were added to 96-well plates seeded with mammalian cells at a ratio of 0.7 infectious viral particles per well to establish clonality. Protocol optimization improved the expression of scFv-Fc and IgGs up to 100μg/mL in 96-well plates, which is sufficient for most antibody characterizations. In addition, 78% of the wells that were positive for protein expression contain only one sequence, indicating successful establishment of clonality in a majority of wells. We have established and optimized a mammalian expression system that produces soluble protein variants in a HTP manner. The system will facilitate developing multiple downstream screening methodologies.

Background: A high quality programmed cell death ligand-1 (PD-L1) diagnostic may help to identify patients (pts) most likely to respond to anti-PD-L1/programmed cell death-1 (PD-1) therapy. Here we describe a PD-L1 immunohistochemical (IHC) diagnostic test developed for urothelial carcinoma (UC) pts treated with durvalumab. Methods: The IHC assay uses an anti-human PD-L1 rabbit mAb optimized for detection of both tumor cell (TC) and tumor-associated immune cell (IC) PD-L1 expression with the OptiView DAB IHC Detection Kit on the automated VENTANA BenchMark ULTRA platform. The assay was validated for intended use in UC formalin-fixed, paraffin-embedded samples in a series of studies that addressed sensitivity, specificity, robustness and precision and implemented in Study CD-ON-MEDI4736-1108 (NCT01693562). Pts were evaluated using the VENTANA PD-L1 (SP263) Assay at a prespecified PD-L1 expression cut-off. Efficacy was analyzed in pts with PD-L1 low/negative (defined as TC Results: The VENTANA PD-L1 (SP263) Assay met all the predefined acceptance criteria (average positive agreement and average negative agreement >85%), showing analytical specificity, sensitivity and precision. It demonstrated ≥97% and ≥85% inter-reader precision agreement for TC and IC respectively. For intra-reader precision, it demonstrated >96% and >87% agreement for TC and IC respectively. For intra-day performance, the assay demonstrated ≥96% agreement for TC and IC and for inter-day performance, it demonstrated ≥98% and 100% agreement for TC and IC respectively. Precision studies for inter-antibody lot, inter-detection kit lot and intra-platform demonstrated >97% agreement for both TC and IC. Inter-laboratory testing was performed at 3 external laboratories and demonstrated an overall agreement rate of 92.3%. The VENTANA PD-L1 (SP263) Assay was implemented in Study CD-ON-MEDI4736-1108 and durvalumab demonstrated clinical activity and durability of response in both PD-L1 high and PD-L1 low/negative subgroups, yet with different response rates. In addition, given the high negative predictive value of the assay, it is especially helpful in evaluating the likelihood of response to durvalumab; pts who were classified as PD-L1 high with the VENTANA PD-L1 (SP263) Assay tended to have a higher objective response rate per RECIST v1.1 than pts who were PD-L1 low/negative. Conclusions: These data show that determination of PD-L1 expression in TC and IC in UC pts using the VENTANA PD-L1 (SP263) Assay is precise and highly reproducible and highlight the utility of the assay in a clinical setting. The VENTANA SP263 Assay is especially helpful in informing pts and physicians on the likelihood of response to durvalumab, but not for the purpose of restricting treatment to only PD-L1 high pts. Citation Format: M Zajac, A M. Boothman, Y Ben, A Gupta, J Antal, X Jin, A Nielsen, G Manriquez, C Barker, P Wang, P Patil, N Schechter, M Rebelatto, J Walker. Analytical validation and clinical utility of an immunohistochemical PD-L1 diagnostic assay for treatment with durvalumab in urothelial carcinoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 664. doi:10.1158/1538-7445.AM2017-664

ADAM17 is the primary sheddase for HER pathway ligands. We report the discovery of a potent and specific ADAM17 inhibitory antibody, MEDI3622, which induces tumor regression or stasis in many EGFR-dependent tumor models. The inhibitory activity of MEDI3622 correlated with EGFR activity both in a series of tumor models across several indications as well in as a focused set of head and neck patient–derived xenograft models. The antitumor activity of MEDI3622 was superior to that of EGFR/HER pathway inhibitors in the OE21 esophageal model and the COLO205 colorectal model suggesting additional activity outside of the EGFR pathway. Combination of MEDI3622 and cetuximab in the OE21 model was additive and eradicated tumors. Proteomics analysis revealed novel ADAM17 substrates that function outside of the HER pathways and may contribute toward the antitumor activity of the monoclonal antibody. Mol Cancer Ther; 14(7); 1637–49. ©2015 AACR.

ADAM17 is the primary sheddase for HER pathway ligands. We report the discovery of a potent and specific ADAM17 inhibitory antibody, MEDI3622, which induces tumor regression or stasis in many EGFR-dependent tumor models. The inhibitory activity of MEDI3622 correlated with EGFR activity both in a series of tumor models across several indications as well as in a focused set of head and neck patient derived xenograft models. Cynomolgus monkey and rat PK/PD assays showed MEDI3622 inhibited TNFα shedding. Toxicity observed in cynomolgus monkey and rat was similar to EGFR inhibitor-induced rash. However, the antitumor activity of MEDI3622 was superior to that of EGFR/HER pathway inhibitors in OE21 head and neck and COLO205 colorectal xenograft models suggesting additional activity outside of the EGFR pathway. Combination of MEDI3622 and cetuximab in the OE21 model was additive and eradicated tumors. Proteomics analysis revealed novel ADAM17 substrates which function outside of the HER pathways and may contribute towards the antitumor activity of the monoclonal antibody. Citation Format: Darrin Sabol, Jonathan RiosDoria, Jon Chesebrough, David Stewart, Kevin Schifferli, Raymond Rothstein, Ching Ching Leow, Jenny Heidbrink-Thompson, Li Cheng, Qun Du, Linda Xu, Xiaofang Jin, Ravinder Tammali, Chanshou Gao, Jay Friedman, Brandy Wilkinson, Melissa Damschroder, Andrew Pierce, MunMun Patnaik, Rong Zeng, Yuling Wu, Susan Spitz, Gabriel Robbie, Lorin Roskos, Robert Hollingsworth, David Tice, Emil Michelotti. Medi3622, a monoclonal antibody to ADAM17, inhibits tumor growth by inhibiting EGFR- and non-EGFR-mediated pathways. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 30. doi:10.1158/1538-7445.AM2015-30